Born in Sublette County, Wyoming, Barlow attended elementary school in a one-room schoolhouse. He was a student at the Fountain Valley School in Colorado. There Barlow met Bob Weir, who would later join the music group the Grateful Dead. Weir and Barlow maintained contact throughout the years; a frequent visitor to Timothy Leary's facility in Millbrook, New York, Barlow introduced the musical group to Leary in 1967. In 1969, Barlow graduated with high honors in comparative religion from Wesleyan University in Middletown, Connecticut, and spent two years traveling. In 1971, he began practicing animal husbandry in Cora, Wyoming, at his family's Bar Cross Land and Livestock Company. He sold that business in 1988.

The seeds of the Barlow-Weir collaboration were sown at a Grateful Dead show at the Capitol Theater in Port Chester, New York, in February 1971. Until this point, Weir had mostly worked with resident Dead lyricist Robert Hunter. Hunter preferred that those who sang his songs stuck to his "canonical" lyrics rather than improvising additions or rearranging words. A feud erupted backstage over a couplet in "Sugar Magnolia" from the band's most recent release (most likely "She can dance a Cajun rhythm/Jump like a Willys in four wheel drive"), culminating in a disgruntled Hunter summoning Barlow and telling him "take him (Weir) -- he's yours." In the fall of 1971, with a deal for a solo album in hand and only two songs completed, Weir and Barlow began to write together for the first time.

Fueled by massive amounts of Wild Turkey and a traditional Native American creativity spell recommended by band friend Rolling Thunder, the twosome hammered out such enduring songs as "Cassidy," "Mexicali Blues," and "Black Throated Wind," all three of which would remain in the repertoires of the Grateful Dead and Weir's varied solo projects for years to come. Other songs to emerge from the Weir-Barlow collaboration include "Let It Grow," "The Music Never Stopped," "Estimated Prophet," "I Need A Miracle," "Lost Sailor," "Saint of Circumstance," and "Throwing Stones." Barlow also did collaborations with Grateful Dead keyboardists Brent Mydland then later Vince Welnick.

In 1986, Barlow joined The WELL online community, then known for a strong Deadhead presence. He served on the company's board of directors for several years. In 1990, Barlow founded the Electronic Frontier Foundation (EFF) along with fellow digital-rights activists John Gilmore and Mitch Kapor. As a founder of EFF, Barlow helped publicize the Secret Service raid on Steve Jackson Games. Barlow's involvement is later documented in the non-fiction book The Hacker Crackdown: Law and Disorder on the Electronic Frontier (1992) by Bruce Sterling. EFF later sponsored the ground-breaking case Steve Jackson Games, Inc. v. United States Secret Service. Steve Jackson Games won the case in 1993.

He married Elaine Parker Barlow, with whom he had three daughters: Amelia Rose, Anna Winter, and Leah Justine. Elaine and John were separated in 1992. He was engaged to Dr. Cynthia Horner, whom he met at a convention center. She died in 1994 from a heart arrhythmia.

Barlow was a good friend of John F. Kennedy Jr.

Barlow is a former chairman of the Sublette County Republican Party and served as campaign manager for Dick Cheney during his 1978 Congressional campaign. By the early 2000s, Barlow was unable to reconcile his ardent libertarianism with the prevailing neoconservative movement and "didn't feel tempted to vote for Bush"; after an arrest for possession of a small quantity of marijuana while traveling, he joined the Democratic Party and publicly committed himself to outright political activism for the first time since his spell with the Republican Party. Barlow has subsequently declared that he is a Republican. He has also claimed on many occasions to be an anarchist. He said in 2004: "I'm embarrassed for my country that in my entire voting life, there has never been a major-party candidate whom I felt I could vote for. All of my presidential votes, whether for George Wallace, Dick Gregory, or John Hagelin, have been protest votes." Barlow said that year he was "voting for John Kerry, though with little enthusiasm."

Barlow currently serves as vice-chairman of the EFF's board of directors. The EFF was designed to mediate the "inevitable conflicts that have begun to occur on the border between Cyberspace and the physical world." They were trying to build a legal wall that would separate and protect the Internet from territorial government, and especially from the U.S. government.

He is a Fellow with the Berkman Center for Internet and Society at Harvard Law School and Diamond Management & Technology Consultants, and a member of the International Academy of Digital Arts and Sciences. He spends much of his time on the road, lecturing and consulting.

Barlow also serves on the advisory boards of Clear Path International, TTI/Vanguard and the global company Touch Light Media founded by Anita Ondine.

From 1971 until 1995, Barlow wrote lyrics for the Grateful Dead, mostly through his relationship with Bob Weir. Amongst others, Barlow's songs include "Cassidy" (about Neal Cassady or Cassidy Law), "Estimated Prophet", "Black-Throated Wind", "Hell in a Bucket", "Mexicali Blues", "The Music Never Stopped", and "Throwing Stones".

His writings include "A Declaration of the Independence of Cyberspace", which was written in response to the enactment of the Communications Decency Act in 1996 as the EFF saw the law as a threat to the independency and sovereignty of cyberspace. He argued that the cyberspace legal order would reflect ethical deliberation instead of the coercive power that characterized real-space governance. Therefore, they found it inappropriate to obtain order in the cyberspace by physical coercion. Instead ethics, enlightened self-interest and the commonwealth were the elements they believed to create a civilization of the Mind in Cyberspace. Later, articles such as "The Economy of Ideas" were also widely circulated in providing a vision for human creativity online.

Barlow has written extensively for Wired magazine, as well as The New York Times, Nerve, and Communications of the ACM. In his writings, he explained the wonder of the Internet. The Internet to him is more than a computer network. It is a place that he called an "electronic frontier". "He frequently wrote in language that echoed Henry Stanley's African diary. "Imagine discovering a continent so vast that it may have no end to its dimensions. Imagine a new world with more resources than all our future greed might exhaust, more opportunities than there will ever be entrepreneurs enough to exploit, and a peculiar kind of real estate that expands with development. Imagine a place where trespassers leave no footprints, where goods can be stolen infinite number of times and yet remain in the possession of their original owners, where business you never heard of can own the history of your personal affairs."" He has wanted to encourage and provoke youngsters to explore the cyberspace through his writing.

Barlow has also returned to writing lyrics, most recently collaborating with the String Cheese Incident's mandolinist and vocalist Michael Kang, including their song "Desert Dawn." Barlow is often seen at String Cheese Incident concerts mixing with the fans and members in the band.

He has also recently collaborated with the Chicago-based jamband Mr. Blotto on their release Barlow Shanghai.