Trivalent arsenite (As(3+)) is a known human carcinogen capable of inducing both cellular transformation and apoptotic cell death by mechanisms involving the production of reactive oxygen species. The tripeptide antioxidant glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. While intracellular levels of GSH are an important determinant of cellular susceptibility to undergo apoptotic cell death, it is not known whether cellular GSH biosynthetic capacity per se regulates As(3+)-induced apoptosis. The rate-limiting enzyme in GSH biosynthesis is glutamate cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. To determine whether increased GSH biosynthetic capacity enhanced cellular resistance to As(3+)-induced apoptotic cell death, we utilized a mouse liver hepatoma (Hepa-1c1c7) cell line stably overexpressing both GCLC and GCLM. Overexpression of the GCL subunits increased GCL holoenzyme formation and activity and inhibited As(3+)-induced apoptosis. This cytoprotective effect was associated with a decrease in As(3+)-induced caspase activation, cleavage of caspase substrates and translocation of cytochrome c to the cytoplasm. In aggregate, these findings demonstrate that enhanced GSH biosynthetic capacity promotes resistance to As(3+)-induced apoptosis by preventing mitochondrial dysfunction and cytochrome c release and highlight the role of the GSH antioxidant defense system in dictating hepatocyte sensitivity to As(3+)-induced apoptotic cell death.