Esculetin is a coumarin derivative present in several plants that have been used as herbal medicine in China for many years. Esculetin has been shown to have a wide spectrum of biologic activities. However, the sulfation of esculetin has not yet been investigated. The present study was designed to identify and characterize the human cytosolic sulfotransferases (SULTs) that have the capacity to sulfate esculetin. A systematic investigation revealed that of the thirteen known human SULTs, SULT1A1, SULT1A3 and SULT1C4 showed the strongest sulfating activity toward esculetin. The pH-dependence and the kinetic parameters of these three SULTs in mediating the sulfation of esculetin were analyzed. HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells were shown to be able to sulfate esculetin under metabolic conditions. In addition, of the four human organ specimens (intestine, kidney, liver, and lung cytosols) examined, liver and intestine cytosols showed considerably higher esculetin-sulfating activity than lung and kidney cytosols. Taken together, these results provided valuable biochemical information for further investigation on the pharmacokinetics of esculetin in vivo.