We present stable oxygen isotope ratio (δ18O) measurements on deep-dwelling planktonic foraminifera from the western margin of the North Atlantic in order to reconstruct the latitude at which the Gulf Stream separated from the western boundary of the Atlantic Ocean in the past. The modern separation latitude can be reconstructed within one degree from δ18O measured on the fossil shells of deep-dwelling planktonic foraminifera Globorotalia truncatulinoides from the Holocene sediments representing the past 10,000 years of mild climate conditions similar to today. The separation latitude is captured in a sharp δ18O gradient, which reflects the prominent hydrographic change across the boundary between the warm waters south and east of the Gulf Stream and the cold Slope Water to the north. The latitudinal δ18O profile from approximately the Last Glacial Maximum shows that the Gulf Stream separated from the coast near Cape Hatteras at almost the same latitude as it does today. We assess our finding in light of existing Gulf Stream separation theories and conclude that one of the theories, the wind-induced separation mechanism, is not consistent with our paleoceanographic reconstruction.