The second part of the 16th century in Paris was largely dominated by what became known as the French Wars of Religion (1562–1598). During the 1520s, the writings of Martin Luther began to circulate in the city, and the doctrines known as Calvinism attracted many followers, especially among the French upper classes. The Sorbonne and University of Paris, the major fortresses of Catholic orthodoxy, fiercely attacked the Protestant and humanist doctrines. The scholar Etienne Dolet was burned at the stake, along with his books, on place Maubert in 1532, on the orders of the theology faculty of the Sorbonne; and many others followed, but the new doctrines continued to grow in popularity.

Henry II was succeeded briefly by Francis II, who reigned from 1559 to 1560; then by Charles IX, from 1560 to 1574, who, under the guidance of their mother, Catherine de Medici, tried at times to reconcile Catholics and Protestants. and at other times, to eliminate them completely. Paris was the stronghold of the Catholic League. On the night of August 23–24, 1572, while many prominent Protestants from all over France were in Paris on the occasion of the marriage of Henri of Navarre—the future Henry IV—to Margaret of Valois, sister of Charles IX, the royal council decided to assassinate the leaders of the Protestants. The targeted killings quickly turned into a general slaughter of Protestants by Catholic mobs, known as St. Bartholomew's Day massacre, and continued through August and September, spreading from Paris to the rest of the country. About three thousand Protestants were massacred by mobs in the streets of Paris, and five to ten thousand elsewhere in France.