Mount Holyoke Female Seminary opened on November 8, 1837 with 116 students. The first graduating class of three students received diplomas on August 23, 1838. Initially, homemaking, teaching and missionary work were the only occupations open to alumnae. By the middle of the nineteenth century, however, increasing numbers of alumnae had entered professions such as social work, librarianship, journalism, medicine, and law. Notable alumnae include poet Emily Dickinson; women's rights advocate Lucy Stone; Olympia Brown, the first woman to become a full-time ordained minister; physician and anesthesiologist Virginia Apgar; playwrights Wendy Wasserstein and Suzan-Lori Parks; Frances Perkins, the first woman to serve as a member of the cabinet of a United States President; and Ella Grasso, the first women elected a state governor in her own right (not as the successor of her husband). A number of these women have been honored on United States postage stamps or with induction into the National Women's Hall of Fame. The Alumnae Association of Mount Holyoke College has also recognized the achievements and contributions of alumnae with a variety of awards including the Alumnae Medal of Honor and the Mary Lyon Award.

Although Mount Holyoke was established as a non-denominational school, most students during the nineteenth century were affiliated with Protestant denominations and the student body was not geographically, racially or ethnically diverse until the mid-twentieth century. The first Native American students, Elinor S. and Mary H. Boudinot (also spelled Boudinott), attended Mount Holyoke in the 1840s and were the daughters of an assimilated Cherokee father. The first known African American student at Mount Holyoke was Hortense Parker, Class of 1883. The first students born outside the United States came to Mount Holyoke from Canada or the Sandwich Islands (now Hawaii) in the 1840s. The first student from Japan, Toshi Miyagawa enrolled in 1889 and the first students from China, Chi Nyok Wang and Ysu Tsit Law, graduated in 1916. Latina and Asian American women began attending Mount Holyoke in the 1930s. In 2010, Mount Holyoke enrolled students from 48 states and nearly 70 countries. On average, approximately one in every three undergraduates is an international citizen or an African American, Asian American, Latina, Native American, or multiracial student.