Giamatti was born in Boston and grew up in South Hadley, Massachusetts, the son of Mary Claybaugh Walton (Smith College '35) and Valentine John Giamatti. His father was professor and chairman of the Department of Italian Language and Literature at Mount Holyoke College. Giamatti's paternal grandparents were Italian immigrants Angelo Giammattei (Italian pronunciation: [dʒammaˈttɛi]) and Maria Lavorgna (Italian pronunciation: [laˈvɔrɲa]): his grandfather Angelo emigrated to the United States from Telese, near Benevento, Italy, around 1900. Giamatti's maternal grandparents, from Wakefield, Massachusetts, were Helen Buffum (Davidson) and Bartlett Walton, who graduated from Phillips Academy Andover and Harvard College.

Giamatti attended South Hadley High School, spent his junior year at the American Overseas School of Rome, and graduated from Phillips Academy in 1956. At Yale College, he was a member of Delta Kappa Epsilon (Phi chapter) and as a junior in 1960 was tapped by Scroll and Key, a senior secret society. He graduated magna cum laude in 1960.

In 1960, he married Toni Marilyn Smith, who taught English for more than 20 years at the Hopkins School in New Haven, Connecticut, until her death in 2004. Together the couple had three children: Hollywood actors Paul and Marcus and jewelry designer Elena. In the film Sideways, a photograph of the character Miles Raymond (portrayed by Giamatti's son, Paul) with his late father is really a picture of Paul and Bart Giamatti.

Giamatti stayed in New Haven to receive his doctorate in 1964, when he also published a volume of essays by Thomas G. Bergin he had coedited with a philosophy graduate student, T. K. Seung. He became a professor of comparative literature at Yale University, an author, and master of Ezra Stiles College at Yale, a post to which he was appointed by his predecessor as Yale President, Kingman Brewster, Jr.

Giamatti taught briefly at Princeton but spent most of his academic life at Yale. Giamatti's scholarly work focused on English Renaissance literature, particularly Edmund Spenser, and relationships between English and Italian Renaissance poets. When Giamatti's tenure as Stiles master ended in 1972, he was so popular that his students wanted to honor him with a present. Giamatti told them he wanted a joke gift and they got him a moosehead (from a yard sale), which was ceremoniously hung in the dining hall.

Giamatti had a lifelong interest in baseball (he was a die-hard Boston Red Sox fan). In 1978, when he was first rumored to be a candidate for the presidency of Yale, he had deflected questions by observing that "The only thing I ever wanted to be President of was the American League." His articles "Tom Seaver's Farewell", published in Harper's Magazine in September 1977, and "Baseball and the American Character," published in that magazine in October 1986, are but two of many of his baseball publications. Giamatti became President of the National League in 1986, and later commissioner of baseball in 1989. During his stint as National League President, Giamatti placed an emphasis on the need to improve the environment for the fan in the ballparks. He also decided to make umpires strictly enforce the balk rule and supported "social justice" as the only remedy for the lack of presence of minority managers, coaches, or executives at any level in Major League Baseball.

Giamatti, whose tough dealing with Yale's union favorably impressed Major League Baseball owners, was unanimously elected to succeed Peter Ueberroth as commissioner on September 8, 1988. Determined to maintain the integrity of the game, on August 24, 1989, Giamatti (who took office on April 1, 1989) prevailed upon Pete Rose to agree voluntarily to remain permanently ineligible to play baseball.

He became the second baseball commissioner to die in office, the first being Kenesaw Mountain Landis. Baseball's owners soon selected Fay Vincent, Giamatti's close friend and baseball's first-ever deputy commissioner, as the new commissioner. On October 14, 1989, before Game 1 at the World Series, Giamatti—to whom this World Series was dedicated—was memorialized with a moment of silence. Son Marcus Giamatti threw out the first pitch before the game. Also before Game One, the Yale Whiffenpoofs sang the national anthem, a blend of "The Star-Spangled Banner" with "America the Beautiful" that has been since repeated by other a cappella groups.

Giamatti was inducted into the National Italian American Sports Hall of Fame in 1992.