The son of Jack MacArthur (an accomplished preacher in his own right), John MacArthur was an athlete and attended Bob Jones University before transferring to Los Angeles Pacific College (now Azusa Pacific University). He later obtained his Masters of Divinity from Biola University's Talbot Theological Seminary, in La Mirada, California. He graduated with honors. From 1964 to 1966, he served as an associate pastor at Calvary Bible Church, in Burbank, California and, from 1966 to 1969, as a faculty representative for Talbot Theological Seminary. Then, in 1969, he became the third pastor in the then-short history of the nondenominational Grace Community Church of Sun Valley, California.

His daily radio program, Grace to You, which is now broadcast throughout much of the world, began as an audio recording ministry to provide cassettes of his sermons to church members who were unable to attend. They were first broadcasted in Baltimore, Maryland, in 1977.

In 1985, MacArthur became the president of The Master's College (formerly Los Angeles Baptist College), an accredited, four-year, liberal arts Christian college; and, in 1986, he founded The Master's Seminary.

MacArthur also received an honorary doctorate from Talbot Theological Seminary and an honorary doctorate from Grace Graduate School.

MacArthur describes himself as a "leaky dispensationalist." While he holds to a premillennial and pre-tribulational rapture of the church and fulfillment of all the covenant promises made to the Jews at the end of the tribulation, he rejects some of the classic dispensational ideas, such as the Law having no application to the church. (He jokingly says that if the tribulation takes place before the Rapture, he'll go straight to anti-Christ headquarters, preach the gospel, and get killed.) Dispensationalism has been a frequent source of controversy; critics claim that MacArthur's deviation from Reformed eschatology has led to an inconsistent Calvinism or self-contradiction suggesting two people groups of God, two roads to salvation, and the so-called, "Great parenthesis."

MacArthur is the person who spear-headed the "Lordship salvation" movement, in the 1980s, that argues against, "Free Grace theology." He states, "you must receive Jesus Christ for who He is, both Lord and Savior, to be truly saved (II Peter 2:20)." Regarding eternal security, he states, "It should never be presented merely as a matter of being once saved, always saved--with no regard for what you believe or do. The writer of states frankly that only those who continue living holy lives will enter the Lord's presence." MacArthur's views raised controversy within American, conservative evangelicalism and were challenged in print by theologians, Charles Ryrie and Zane C. Hodges, who argued that MacArthur was teaching a form of works-based salvation.

In December 1989, the Bible Broadcasting Network terminated MacArthur's "Grace to You" program. In explaining that step, BBN president Lowell Davey referred to MacArthur's teachings on the blood of Christ, "Lordship Salvation," and, "Hyper-Calvinism." Davey called these teachings, "confusing." In a letter dated January 15, 1990, Davey cited a, "....drift by Dr. MacArthur to a theological position that we could not adhere to," and said that MacArthur's sermon series on the theology of election, "....convinced us that the direction of 'Grace to You' was toward Hyper-Calvinism...." MacArthur preaches "Salvation" by election of God's sovereignty; he accepts Arthur Pink's view of the sovereignty of God in salvation. However, the term "Hyper-Calvinism" is used by some to denote 5-point Calvinism or even any strong defense of Calvinism, rather than the historical "Hyper-Calvinism" position that only the "Elect" may be offered the Gospel. This position does not seem to reflect the truth of MacArthur's position in his sermons. The controversy concerning the efficacy of the Christ's blood stems from MacArthur's statement that it is not the literal liquid blood of Christ that saves, but his sacrificial death on the Cross, a view that he espoused in an article titled, "Not His Bleeding, but His Dying," published in the May 1976 issue of the Grace to You family paper that is distributed to his church.

In 1983, MacArthur first published his belief in the doctrine of "incarnational sonship." In 1989, after some criticism, he defended his views in a plenary session of the annual convention of the Independent Fundamental Churches of America. Subsequently, MacArthur has written that he has reversed this position and no longer regards Christ's sonship as a role he assumed in his incarnation.

MacArthur is a cessationist, arguing that Charismatic and Pentecostal theology and their practice regarding the "gifts of the Spirit" (healing, miracles, Speaking in Tongues, etc.) are not for these times. Rich Nathan, pastor of Vineyard Church of Columbus published "A Response to Charismatic Chaos" in 1993.

His writings are similarly critical of other modern Christian movements such as those who run "seeker-friendly" church services such as Robert Schuller, Bill Hybels, and Rick Warren. He is also an advocate of Biblical Counseling (also known as Nouthetic Counseling), which stresses the Bible as a tool for counseling and rejects psychological theories and techniques, considering Psychology as contrary to the Bible. His stance has caused several controversies, the most notable of which was the first time an employee of an evangelical church had ever been sued for malpractice.

He has called Catholicism "a Satanic religious system that wants to engulf the earth." John MacArthur does not believe that Roman Catholics are Christians, and believes that ecumenism will not work because it is "another religion". He said that Roman Catholic "Priests are broken, shattered, tragic, sad, disconnected people; no past, no present, no future. They belittle the sanctity of the marriage relationship. They are denied normal relationships; the friendship of marriage. They are victims of a terrible system with no biblical basis whatsoever. It is a soul-destroying process that leaves them in a situation of rampant temptation, exposure to the worst. And the only way to fulfill these drives is sinfully. And they do not have the restraint of a transformed or sanctified life." Regarding monks, MacArthur says "the inmates, I call them inmates, of monasteries, are unmarried men. Its just bizarre and abnormal. I read today the 50% are homosexual when they get there. The rest have no chance. These people are predators." He says of convents, "I am surprised Amnesty International doesn't raid these places."

MacArthur has stated that the "theology of Islam is false," and that Allah is the "wrong god."

MacArthur has also been a proponent of Christian abstinence from alcohol, though he does not think it is necessarily sinful. (See Christianity and alcohol.)