Homocysteine (Hcy) and glutathione (GSH) are crucial reductionoxidation mediators. The underlying mechanisms governing the effects of Hcy on GSH generation in the progression of alcoholic liver disease has so far received little attention. The present study hypothesized that the antioxidant transcriptional factor nuclear factor (erythroidderived 2)like 2 (Nrf2) may participate in Hcymediated regulation of GSH production in HepG2 human liver cancer cells. MTT assay was used to study the cytotoxicity of homocysteine, western blot analysis and immunofluorescence staining were used to determine the effect of Hcy on Nrf2 expression. Our data demonstrated that HepG2 cells exposed to exogenous levels of Hcy (0100 microM) exhibited elevated GSH levels in a concentrationdependent manner. Furthermore, 4hydroxynonenal (4HNE)induced cell injury was attenuated by Hcy; however, this protective effect was blocked by the GSHproduction inhibitor buthionine sulfoximine. Hcy treatment was able to induce Nrf2 protein expression in HepG2 cells. Treatment with the Nrf2 activator tertbutylhydroquinone (0100 microM) increased GSH expression in a concentrationdependent manner; however, Nrf2siRNA abolished the Hcyinduced increase in GSH expression and cellular protection in 4HNEstressed HepG2 cells. In conclusion, the antioxidant transcriptional factor Nrf2 was demonstrated to mediate the Hcyinduced increase in GSH expression levels and cellular protection in HepG2 cells.