Background. The nephrotoxicity induced by immunosuppressant FK506 remains a serious clinical problem, and the underlying mechanism has not been completely understood. The present study was undertaken to determine the role of hydrogen peroxide in FK506-mediated cytotoxicity in a porcine renal proximal tubular cell line, LLC-PK1 cells, and human embryonic kidney (HEK293) cells. Methods. Cytotoxicity was estimated by crystal violet and lactate dehydrogenase release assays. The activity of reactive oxygen species (ROS) was detected by flow cytometry. FK506-induced cell death was examined in the presence of the hydrogen peroxide scavenger, catalase, or a scavenger of hydroxyl radicals, sodium benzoate. As a control, FK506-induced cell death was also measured in the presence of superoxide anion inhibitor, 4,5-dihydroxy-1,2-benzene disulfonic acid (Tiron), TEMPO, or overexpressed human manganese superoxide dismutase (MnSOD). Catalase was also used in tumor necrosis factor-α (TNF-α)-induced cell injury to determine whether the enzyme specifically protected cells against FK506-mediated cytotoxicity. Results. FK506 induced cell death in a dose-dependent manner and coincided with a dose-dependent increase in ROS activity. Abrogation of FK506-mediated ROS by catalase and N-acetylcysteine blunted FK506-induced cell death. Furthermore, overexpression of catalase, sodium benzoate, and deferoxamine inhibited the cytotoxic effect of FK506. In contrast, Tiron, TEMPO, or overexpression of human MnSOD failed to show cytoprotection. In fact, TEMPO or expression of MnSOD enhanced the effect of FK506. Catalase did not significantly affect TNF-α-induced cell injury. Conclusion. Catalase is uniquely required in cellular protection against FK506 cytotoxicity, which suggests an important role for hydrogen peroxide in the cellular actions of FK506.