Holsti worked at Stanford University as an instructor in the Department of Political Science (1962–1965), the research coordinator and associate director of Studies in International Conflict and Integration (1962–1967) and assistant professor in the Department of Political Science, (1965–1967). He moved to the University of British Columbia in 1967, working as assistant professor in the Department of Political Science (1967–1971) before becoming a professor (1971–1974).
Holsti was at the University of California, Davis, as a professor in the Department of Political Science (1978–1979) before joining the faculty of Duke University as George V. Allen Professor in the Department of Political Science (1974–1998). He became a professor emeritus in 1998.
Holsti's brother Kal is also a political scientist. Their father, Rudolf Holsti, served as Finland's foreign minister.