MacKinnon was born in Minnesota. Her mother is Elizabeth Valentine Davis; her father, George E. MacKinnon was a lawyer, congressman (1947 to 1949), and judge on the U.S. Court of Appeals for the D.C. Circuit (1969 to 1995). She has two younger brothers.
MacKinnon became the third generation of her family to attend her mother's alma mater, Smith College. She graduated in the top 2% of her class at Smith and earned a J.D. and a Ph.D. from Yale University. While at Yale Law School, she received a National Science Foundation fellowship.