In an 1835 pamphlet outlining her plans for Mount Holyoke Female Seminary, Mary Lyon stated that one of the goals of the school would be "to cultivate the missionary spirit among its pupils." Soon after the Seminary opened on November 8, 1837 alumnae began serving as missionaries in the United States and in other countries, where they opened and staffed influential schools, brought aid to orphans and others, and supported social reforms to improve the lives of girls and women. Alumnae were affiliated with the American Board of Commissioners for Foreign Missions (ABCFM), a Congregational Church organization founded in 1810, or with the missionary boards of other Protestant denominations. The first alumna missionary was Charlotte Bailey (1838), who worked with her husband, Aldin Grout, among the Zulus in South Africa. Other notable foreign missionaries during the nineteenth century were Fidelia Fiske (1842, Persia); Lyon's nieces Abigail Moore Burgess (1838, India) and Lucy Lyon Lord (1840, China); Charlotte and Mary Ely (1861, Armenia); Alice Gordon Gulick (1867, Spain); and Mary Otis Preston Spafford (1879, South Africa). Alumnae were also missionaries to the Cherokee and other Native Americans and to former African American slaves. Members of the Mount Holyoke community actively supported this work by raising funds for alumnae missionaries and establishing a number of organizations for students interested in missionary service, including the Student Volunteer Band and the College's chapter of the Young Women's Christian Association. Many missionaries and officials from mission boards also presented lectures and programs at Mount Holyoke.

The missionary service of alumnae continued in the twentieth century, exemplified by the work of Alice Browne Frame (1900) in China, Charlotte Allen Ward (1903) in Lebanon and Turkey, Ruth Parker White (1917) in India, Katharine Merrill (1917) in Japan, and Leila Childs Edling (1917) in Angola. Elsie Kimball (1909) and numerous other alumnae supported Near East Relief efforts in Europe, the Middle East, and Asia during and after World War I, while Grace Bacon (1918), Florence Brugger (1922), and many other Mount Holyoke women worked for the American Red Cross and other organizations during both world wars. Beginning in the 1960s, Gena Lee Reisner (1964) and other alumnae demonstrated the College's missionary spirit by joining the Peace Corps, Teach for America, and other initiatives designed to bring education and health care to those in need. Mount Holyoke students such as Sandra Nichols Ward (1965) engaged in this work as undergraduates by participating in Operation Crossroads Africa and similar programs.