The endocannabinoid system is an important regulator of hepatic fibrogenesis. In this study, we determined the effects of 2‐arachidonoyl glycerol (2‐AG) on hepatic stellate cells (HSCs), the main fibro‐genic cell type in the liver. Culture‐activated HSCs were highly susceptible to 2‐AG‐induced cell death with >50% cell death at 10 μM after 18 h of treatment. 2‐AG‐induced HSC death showed typical features of apoptosis such as PARP‐ and caspase 3‐cleavage and depended on reactive oxygen species (ROS) formation. Confocal microscopy revealed mitochondria as primary site of ROS production and demonstrated mitochon‐drial depolarization and increased mitochondrial permeability after 2‐AG treatment. 2‐AG‐induced cell death was independent of cannabinoid receptors but required the presence of membrane cholesterol. Primary hepatocytes were resistant to 2‐AG‐induced ROS induction and cell death but became susceptible after GSH depletion suggesting antioxidant defenses as a critical determinant of 2‐AG sensitivity. Hepatic levels of 2‐AG were significantly elevated in two models of experimental fibrogenesis and reached concentrations that are sufficient to induce death in HSCs. These findings suggest that 2‐AG may act as an antifibrogenic mediator in the liver by inducing cell death in activated HSCs but not hepatocytes.—Siegmund, S. V., Qian, T., de Minicis, S., Harvey‐White, J., Kunos, G., Vinod, K Y., Hungund, B., Schwabe, R. F. The endocannabinoid 2‐arachidonoyl glycerol induces death of hepatic stellate cells via mitochondrial reactive oxygen species. FASEB J. 21, 2798–2806 (2007)