MacKinnon was born in Minneapolis, Minnesota, the first of three children (a girl and two boys) to Elizabeth Valentine Davis and George E. MacKinnon; her father was a lawyer, congressman (1947–1949), and judge on the U.S. Court of Appeals for the D.C. Circuit (1969–1995).
She became the third generation of her family to attend her mother's alma mater, Smith College, graduating in the top two percent of her class. She obtained her J.D. from Yale University in 1977 and a PhD in political science, also from Yale, in 1987. While at Yale, she received a National Science Foundation fellowship.