AbstractCinnabar is a natural mercury sulfide (HgS) mineral of volcanic or hydrothermal origin that is found worldwide. It has been mined prehistorically and historically in China, Japan, Europe and the Americas to extract metallic mercury (Hg⁰) for use in metallurgy, as a medicinal, a preservative and as a red pigment for body paint and ceramics. Processing cinnabar via combustion releases Hg⁰ vapor that can be toxic if inhaled. Mercury from cinnabar can also be absorbed through the gut and skin, where it can accumulate in organs and bone. Here, we report moderate to high levels of total mercury (THg) in human bone from three Late Neolithic/Chalcolithic (5400–4100 B.P.) sites in southern Portugal that were likely caused by cultural use of cinnabar. We use light stable isotope and Hg stable isotope tracking to test three hypotheses on the origin of mercury in this prehistoric human bone. We traced Hg in two individuals to cinnabar deposits near Almadén, Spain and conclude that use of this mineral likely caused mild to severe mercury poisoning in the prehistoric population. Our methods have applications to bioarchaeological investigations worldwide and for tracking trade routes and mobility of prehistoric populations where cinnabar use is documented.