We previously demonstrated that albumin increases intracellular glutathione (GSH) levels in human astrocytoma U-251 MG cells. GSH has been known to play an important in oxidative stress tolerance and anticancer drug resistance in tumor cells. In this study, we investigated the effect of albumin on hydrogen peroxide- and nimustine (ACNU)-induced cell death in U-251 MG cells using bovine serum albumin (BSA). Intracellular GSH levels were determined by the DTNB recycling assay. Cell death of U-251 MG cells was examined by Hoechst 33342/Propidium iodide staining. Treatment of U-251 MG cells with BSA (0.1-10 mg/mL) increased the levels of intracellular GSH and catalytic subunit of glutamate-cysteine ligase (GCLc), a rate-limiting enzyme in GSH synthesis, protein in a concentration-dependent manner. In addition, pretreatment with BSA for 24 h attenuated hydrogen peroxide (0.75 mM)- and ACNU (1 mM)-induced cell death in a concentration-dependent manner. This cytoprotective effect of BSA was not observed in the presence of DL-Buthionine-(S,R)-sulfoximine (1 mM), an inhibitor of glutathione synthesis. These results suggest that albumin increases intracellular GSH levels in U-251 MG cells by inducing GCLc protein and that albumin suppresses hydrogen peroxide- and ACNU-induced cell death via increasing intracellular GSH levels.