Born in Rochester, New York, to a wealthy Jewish Bostonian family, he was educated at Harvard, from which he was graduated in 1930. His father was chairman of Filene's Department Store, in Boston, and his mother was the daughter of a successful clothing manufacturer in Rochester, New York.

His interest in ballet and George Balanchine started when he saw Balanchine's Apollo performed by the Ballet Russe. He became determined to get Balanchine to America. Together with Edward M. M. Warburg (a classmate from Harvard), they started the School of American Ballet in Hartford, Connecticut, in October 1933. The studio moved to the fourth floor of a building at Madison Avenue and 59th Street in New York City in 1934. Warburg's father invited the group of students from the evening class to perform at a private party. The ballet they did was "Serenade", the first major ballet choreographed by Balanchine in America. Just months later Kirstein and Warburg founded, together with Balanchine and Dimitriev, the American Ballet.

This became the resident company of the Metropolitan Opera. That arrangement was unsatisfactory because the Opera would not allow Balanchine and Kirstein artistic freedom.

His career was interrupted by the United States' entry into World War II. In 1943, he enlisted; and he was assigned to the U.S. Third Army. Private First Class Lincoln Kirstein He was a Monuments, Fine Arts, and Archives (MFAA) Officer..

In 1946, Balanchine and Kirstein founded the Ballet Society, renamed the New York City Ballet in 1948. He served as the company's General Director from 1946 to 1989.

Kirstein wrote in a 1959 monograph called "What Ballet Is All About":

"Our Western ballet is a clear if complex blending of human anatomy, solid geometry and acrobatics offered as a symbolic demonstration of manners...the morality of consideration for one human being moving in time with another."

Kirstein's eclectic interests, ambition and keen interest in high culture, funded by independent means, drew a large circle of friends who stimulated creativity in many of the arts. These included: Glenway Wescott, Monroe Wheeler, George Platt Lynes, Jared French, Bernard Perlin, Pavel Tchelitchev, Katherine Anne Porter, Barbara Harrison, Gertrude Stein, Jensen Yow, Jonathan Tichenor, Cecil Beaton, Jean Cocteau, George Tooker, Margaret French, Walker Evans and more.

He was married in 1940 to Fidelma Cadmus. While he and his wife enjoyed an amicable relationship, Kirstein also continued to pursue affairs with men. The New York art world considered his bisexuality an "open secret," although he did not publicly acknowledge his sexual orientation until 1982. A recent biography about his life has explored his relationship with Soviet filmmaker Sergei Eisenstein.

Kirstein was the primary patron of the artist Paul Cadmus, Fidelma's brother. He purchased many of his paintings and subsidized his living expenses. Cadmus had difficulty selling his work through galleries because of the erotically charged depictions of working and middle class men, which provoked great controversy.

English critic Clement Crisp wrote:...

"He was one of those rare talents who touch the entire artistic life of their time. Ballet, film, literature, theatre, painting, sculpture, photography all occupied his attention."

Kirstein helped organize a 1959 American tour for of musicians and dancers from the Japanese Imperial Household Agency. At that time, Japanese Imperial court music gagaku had only rarely been performed outside the Imperial Music Pavilion in Tokyo at some of the great Japanese shrines.

Kirstein commissioned and helped to fund the physical home of the New York City Ballet: the New York State Theater building at Lincoln Center, designed in 1964 by architect Philip Johnson (1906—2005). Despite its conservative modernist exterior, the glittery red and gold interior recalls the imaginative and lavish backdrops of the Ballets Russes. He served as the general director of the ballet company from 1948 to 1989.

Kirstein's and Balanchine's collaboration lasted until the latter's death in 1983. On March 26, 1984, President Ronald Reagan presented Kirstein with the Presidential Medal of Freedom for his contributions to the arts.

Kirstein was a serious collector. Early in the history of the Dance Collection, he gave the New York Public Library for the Performing Arts a wealth of rare dance materials. Before his death in 1996, Kirstein donated all his papers, artworks, and other materials related to the history of dance and his life in the arts to the Dance Collection. These treasures in the Kirstein collection will inform future generations' pursuing the knowledge of dance.