The St. Lawrence estuary was visited by many navigators (such as John Cabot and Jacques Cartier) and Basque fishermen soon after the discovery of America (or perhaps even before). But the first known European explorer to sail the inland part of the St. Lawrence was Jacques Cartier, during his second trip to Canada in 1535, with the help of Iroquoian chief Donnacona's two sons. As he arrived in the estuary on St. Lawrence's feast day, Cartier accordingly named it the Gulf of St. Lawrence.[4] The land along the river was inhabited at the time by the St. Lawrence Iroquoians. The St. Lawrence River is partly with the U.S. and as such is that country's sixth oldest surviving European place-name.

Until the early 1600s, the French used the name Rivière du Canada to designate the Saint Lawrence upstream to Montreal and the Ottawa River after Montreal. The Saint Lawrence River served as the main route for European exploration of the North American interior, first pioneered by French explorer Samuel de Champlain.
Because of the virtually impassable Lachine Rapids, the St. Lawrence was once continuously navigable only as far as Montreal. Opened in 1825, the Lachine Canal was the first to allow ships to pass the rapids. An extensive system of canals and locks, known as the Saint Lawrence Seaway, was officially opened on 26 June 1959 by Queen Elizabeth II (representing Canada) and President Dwight D. Eisenhower (representing the United States of America). The Seaway now permits ocean-going vessels to pass all the way to Lake Superior.
During World War II, the Battle of the St. Lawrence involved a number of submarine and anti-submarine actions throughout the lower St. Lawrence River and the entire Gulf of Saint Lawrence, Strait of Belle Isle and Cabot Strait from May to October 1942, September 1943, and again in October and November 1944. During this time, German U-boats sank a number of merchant marine ships and three Canadian warships.
In the late 1970s, the river was the subject of a successful ecological campaign (called "Save the River"), originally responding to planned development by the United States Army Corps of Engineers. The campaign was organized, among others, by Abbie Hoffman, then on the run under the pseudonym of Barry Freed.