Hydrogen peroxide (H₂O₂) induces the hypertrophy in cultured H9c2 cardiomyocytes and cell death in glutathione (GSH)-depleted H9c2 cells. In the present study, we observed that pretreatment with a serine protease inhibitor, N-a-tosyl-L-lysine chloromethyl ketone (TLCK), significantly prevented the H2O2-induced cell damages in GSH-depleted H9c2 cells in a concentration-dependent manner. The phase contrast microscopy revealed that although the exposure of the GSH-depleted H9c2 cells to H₂O₂ resulted in a globular shape of the cells, TLCK prevented the occurrence of H₂O₂-induced morphological changes. TLCK also inhibited the generation of reactive oxygen species in the cells after addition of H₂O₂, suggesting that the antioxidant action of TLCK is involved in the protection against the cell damages by H₂O₂. Application of TLCK after ~30 min of exposure to H₂O₂ could significantly protect the cells from cell damages. The other serine protease inhibitors that were tested could not prevent the cell damages in GSH- depleted H9c2 cells. Pretreatment with an inhibitor of nuclear factor-kB translocation into the nucleus and a proteasome inhibitor did not prevent the cell damages in GSH-depleted H9c2 cells. An inhibitor of p53 significantly prevented the cell damages in GSH-depleted H9c2 cells. These results suggest that antioxidative action of TLCK prevents the death of GSH-depleted H9c2 cardiomyocytes induced by H₂O₂.