Born in Sommerhausen, Franconia, to a prosperous Lutheran family, he was trained as a lawyer in some of the best German universities of his day, including the University of Altdorf, the University of Strasbourg, and the University of Jena. He started his practice in Windsheim and Frankfurt-am-Main. He was a close friend of the German Pietist leader Philipp Jakob Spener during the early development of Spener's movement in Frankfurt. From 1680 through 1682, he worked as a tutor accompanying a young nobleman during his Wanderjahr through Germany, England, France, Switzerland and Holland.
In 1683, a group of Mennonites, Pietists, and Quakers in Frankfurt approached Pastorius about acting as their agent to purchase land in Pennsylvania for a settlement. Pastorius took passage to Philadelphia. There he negotiated the purchase of 15,000 acres (61 km²) from William Penn, the proprietor of the colony, and laid out the settlement of Germantown, where he himself would live until his death.
As one of Germantown's leading citizens, Pastorius served in many public offices and wrote extensively on topics ranging from beekeeping to religion. He was also a skilled poet whose work appears in the New Oxford Book of Seventeenth-Century Verse (ISBN 0-19-214164-3). Though raised as a Pietist Lutheran, he grew close to Quakerism. In 1688, he and three Germantown Quakers joined in signing a Memorial Against Slaveholding, the first one made in the English colonies.
Also in 1688, Pastorius married Ennecke Klostermanns. They had two sons. Pastorius died sometime between December 26, 1719, and January 13, 1720. His life was celebrated by the Quaker poet John Greenleaf Whittier in The Pennsylvania Pilgrim.
Despite the Quaker sympathies of Pastorius, his name was appropriated in 1942 by the Abwehr of Nazi Germany for "Operation Pastorius," a failed sabotage attack on the United States in World War II that included a target in Philadelphia.