Carlisle's early education took place in New Orleans. In 1921, she was taken to Europe, where her mother hoped to marry her off to European royalty, believing the nobility there more amenable to a Jewish bride – only to end up flitting around Europe and often living in what Carlisle recalled as "the worst room of the best hotel." Carlisle was educated at the Chateau Mont-Choisi in Lausanne, Switzerland, then at the Sorbonne and the London School of Economics. She studied acting in London at the Royal Academy of Dramatic Art.

After returning to New York in 1932 with her mother, she appeared, billed as Kitty Carlisle, on Broadway in several operettas and musical comedies, and in the American premiere of Benjamin Britten's The Rape of Lucretia. She also sang the title role in Georges Bizet's Carmen in Salt Lake City. She privately studied voice with Juilliard Teacher Anna E. Schoen-Rene, who had been a student of Pauline Viardot-Garcia and Manuel Garcia.

Carlisle's early movies included Murder at the Vanities (1934), A Night at the Opera (1935) with the Marx Brothers, and two films with Bing Crosby, She Loves Me Not (1934) and Here Is My Heart (1934). Carlisle resumed her film career later in life, appearing in Woody Allen's Radio Days (1987) and in Six Degrees of Separation (1993), as well as on stage in a revival of On Your Toes, replacing Dina Merrill. Her last movie appearance was in Catch Me If You Can (2002) in which she played herself in a dramatization of a 1970s To Tell the Truth episode.

Late in 1933, Kitty Carlisle dated George Gershwin, "until George went to California". Carlisle married Playwright and theatrical Producer Moss Hart on August 10, 1946, the two having met as actors at the Bucks County Playhouse in New Hope, Pennsylvania. The couple had two children. Hart died on December 20, 1961, at their home in Palm Springs, California. Carlisle never remarried, but briefly dated former governor and presidential candidate Thomas E. Dewey after the death of his wife.

In the early 1950s, Carlisle was an occasional panelist on the NBC game show, Who Said That?, in which celebrities try to determine the speaker of quotations taken from recent news reports.

Carlisle became a household name through To Tell the Truth, where she was a regular panelist from 1956 to 1978, and later appeared on revivals of the series in 1980, 1990–91 and one episode in 2000. (One of her most notable hallmarks was her writing of the number one: When she voted for the member of the team of challengers who occupied the number one seat, it was written with a Roman numeral I.) She was also a semi-regular panelist on Password, Match Game, Missing Links, and What's My Line?

For her contributions to the film industry, Carlisle was inducted into the Hollywood Walk of Fame in 1960 with a motion pictures star located at 6611 Hollywood Boulevard.

On December 31, 1966, Carlisle made her debut with the Metropolitan Opera, as Prince Orlofsky in Strauss's Die Fledermaus. She sang the role 10 more times that season, then returned in 1973 for four more performances. Her final performance with the company was on July 7, 1973. She reprised this role during the Beverly Sills Farewell Gala in October 1980.

Carlisle Hart was a longtime champion of Historic Preservation in New York City and State. While chair of the New York State Council on the Arts (NYSCA) from 1976-1996, Mrs. Hart directed many millions of dollars in support to preservation projects from the Niagara Frontier to Staten Island in an effort to keep historic preservation as a core program of the New York State Council on the Arts, the only arts council in America that provides such funding. In 1980, she was crowned Queen of the Beaux Arts Ball, an annual event run by the Beaux Arts Society (Paul Lynde was coronated King the same year). The Egg, a notable Mid-century modern structure at Albany, New York's Empire State Plaza, was also renamed in her honor.

During the 1980s and 1990s, Kitty Hart was the partner of diplomatic Historian Ivo John Lederer, and their relationship lasted 16 years until Lederer's death in 1998. In her later years, she kept company with the financier and art collector Roy Neuberger. She also widely performed her one-woman show in which she told anecdotes about the many great men in American musical theatre history whom she had personally known, notably George Gershwin who had proposed marriage (according to an interview in American Heritage magazine), Irving Berlin, Kurt Weill, Oscar Hammerstein, Alan Jay Lerner, and Frederick Loewe, interspersed with a few of the songs that made each of them famous.

In recognition of this legacy, the Historic Districts Council bestowed its Landmarks Lion award upon her in 2003.

In 2006, Carlisle performed at Feinstein's at the Regency in New York City; in St. Louis, Missouri; Phoenix, Arizona; Atlanta, Georgia; and at the famed Plush Room in San Francisco. According to her official website, her appearances in Atlanta in November 2006 were her last public performances. In December 2006, she made her final public appearance as the special Celebrity guest for the annual Noël Coward Society birthday tribute in which she laid flowers in front of Coward's statue at The Gershwin Theatre in New York City.

Carlisle died on April 17, 2007, from congestive heart failure resulting from a prolonged bout of pneumonia. She had been in and out of the hospital since she contracted pneumonia some time prior to November 2006. She died in her Manhattan apartment, with her son, Christopher Hart, at her bedside. She was interred in a crypt next to her husband, Moss Hart, at Ferncliff Cemetery in Hartsdale, New York.