Mamoulian was born in Tiflis (Georgia) to an Armenian family with roots in Lori and Nakhichevan. His father Zakaria was a former member of the Russian military and a banker. His mother Vergine Kalantarian-Mamoulian had been president of the Armenian Dramatic Society of Tiflis and an amateur actress on the Armenian scene. The Mamoulians spoke Russian at home, but Rouben learned to speak and to write Armenian at an early age. His primary education was at the Lycée Montaigne of Paris, where future French director René Clair was one of his classmates. Later, he continued his studies at the Russian high school of Tiflis and the School Law of Moscow University, while he also took classes at the theatrical courses opened by Armenian director Evgeni Vakhtangov at the Art Theater. He returned to Tiflis in 1918, where he opened a theatrical studio with Levon Kalantar and Suren Khachaturian, and published theatrical reviews in Russian and Armenian newspapers. He also wrote poetry in Russian.
Mamoulian moved to England in 1920, where his sister Svetlana was married to a Scottish soldier. He first directed Austin Page’s play The Beating on the Door in November 1922. Favorable reviews in the London press made him known in France and the United States. Russian opera singer Vladimir Rosing brought him to America the next year to teach at the newly founded Eastman School of Music (created by George Eastman, the founder and owner of Kodak Company) in Rochester (New York) and was involved in directing opera and theatre in 1924-1925.
In 1925, Mamoulian and American modern dancer Martha Graham together produced a short two-color film called The Flute of Krishna, featuring Eastman students. They both left the school shortly thereafter. He began his Broadway director career in October 1927 with a production of DuBose Heyward’s Porgy, which made his international fame at once for its characteristics. He directed the revival of Porgy in 1929 along with George Gershwin’s operatic treatment, Porgy and Bess (1935). He was also the first to stage such notable Broadway works as Oklahoma! (1943), Carousel (1945), and Lost in the Stars (1949).
His first feature film, Applause (1929), was one of the earliest talkies. It was a landmark film owing to Mamoulian's innovative use of camera movement and sound. These qualities were carried through to his other films released in the 1930s. Dr. Jekyll and Mr. Hyde (1931) considered the best version of Robert Louis Stevenson’s tale, benefited from having been made before the Production Code came into full force, the same as Queen Christina (1933), the last film that Greta Garbo made with John Gilbert.
He directed the first three-strip Technicolor film, Becky Sharp (1935), based on William Thackeray’s novel Vanity Fair, as well as the 1937 musical High, Wide, and Handsome. His next two films, The Mark of Zorro (1940) and Blood and Sand (1941), starred Tyrone Power. These were remakes of silent movies that earned him wide admiration. Blood and Sand used color schemes based on the work of Spanish artists such as Diego Velázquez and El Greco. His foray into screwball comedy genre was a success: Rings on Her Fingers (1942) starred Henry Fonda and Gene Tierney.
In the early 1930s the Armenian film studio Armenfilm negotiated with him to film a movie in Soviet Armenia, which never happened. Mamoulian was going to direct the 1935 Metro Goldwyn Mayer version of The Forty Days of Musa Dagh, which was stopped due to Turkish pressure on the U.S. State Department.
Mamoulian was recruited in 1936 by King Vidor, co-founder of Directors Guild of America (DGA), to help unionize fellow movie directors. His lifelong allegiance to the DGA, as well as his general unwillingness to compromise, contributed to his being targeted in Hollywood’s blacklisting of the 1950s. His last completed musical film was the 1957 version of the Cole Porter musical Silk Stockings, with Fred Astaire and Cyd Charisse. His film directing career came to an end when he was consecutively fired from two movies, Porgy and Bess (1959) and Cleopatra (1963).
He lived and created outside the Armenian milieu, but he was frequently in contact one way or another. In an event in his honor held in 1932 at the Nubarian Library of Paris, he noted: “The fact that I have not forgotten my being Armenian, despite living in a foreign environment, is very natural; the contrary would be unnatural.” He rejected all proposals to have his name changed or shortened, at a time when ethnic surnames were not so favored in America.
In the summer of 1971 Mamoulian visited Soviet Armenia, but except for a quick visit to the main tourist attractions, he did not enjoy any official reception and journalists were banned from meeting him. The reason was that he had been denounced as an enemy of the Soviet Union for having filmed an anti-Soviet movie (Silk Stockings, where the KGB spy Ninotchka did not return to the Soviet Union).
Mamoulian was inducted into the American Theater Hall of Fame in 1981 and received a Lifetime Achievement Award from the Directors Guild of America in 1982. He has a star on the Hollywood Walk of Fame.
He was not only a director, but also a theoretician of films and theater. He wrote many articles on these issues. He also wrote screenplays, shorts stories, and a version of Hamlet in contemporary English. He died on December 4, 1987 of natural causes in Woodland Hills, California, at the age of 90. The funeral services were held at the St. Mary Armenian Apostolic Church of Glendale and the Armenian director was buried at the Forest Lawn cemetery of Glendale. His wife, painter Azadia Newman, died in 1999 at the age of 97.