He was born in Gifford, Scotland and obtained a Master of Arts at Edinburgh, and went on to divinity school. He became a Presbyterian minister at Beith, where he married and wrote three well-known works on theology. He was awarded a Doctorate of Divinity from the University of St. Andrews.
At the urging of Benjamin Rush and Richard Stockton, he and his wife emigrated to New Jersey in 1768, where he took up the position of President of the College of New Jersey (which was later to become Princeton University).
He came to support the Revolution and joined the committees of correspondence and safety in early 1776. He was elected to the Continental Congress and voted for the Resolution for Independence. In answer to an objection that the country was not yet ready for independence, he replied that it "was not only ripe for the measure, but in danger of rotting for the want of it."
In November, 1788, as British forces neared, he closed and evacuated the College of New Jersey. The buildings were nearly destroyed and Witherspoon was responsible for its rebuilding after the war. He served twice in the New Jersey Legislature .
He had a series of eye injuries and was blind when he died on his farm "Tusculum" just outside of Princeton.