Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most potent anticancer drugs. However, the therapeutic value of CDDP is greatly compromised by its dose-limiting nephrotoxicity. This study was performed to investigate whether reduced glutathione (GSH) was able to reduce the kidney injury induced by CDDP and whether it affected the anticancer activity of CDDP in vivo and in vitro. In in vivo experiments, mice were divided into five groups: control, CDDP only and three GSH treatment groups. Blood samples were collected 72 h after CDDP administration to determine the levels of blood urea nitrogen (BUN) and plasma creatinine (Cr). In addition, we examined antioxidative parameters, malondialdehyde (MDA) levels and histopathological changes in the kidney. In order to investigate whether GSH affected the anticancer activity of CDDP, we performed a sulforhodamine B (SRB) assay to determine the anti-proliferative effect in three tumor cell lines of treatment with CDDP alone or combined with GSH and examined the cell morphology. The results revealed that GSH decreased the BUN and Cr levels in plasma, ameliorated the pathological changes induced by CDDP and enhanced the endogenous antioxidant capacities in all three GSH groups. Furthermore, GSH significantly inhibited the growth of the three tumor cell lines when combined with CDDP and did not affect the inhibitory effect of CDDP on the carcinoma cell proliferation. In addition, we found no differences among the three GSH groups. These findings suggest that GSH is able to attenuate the nephrotoxicity induced by CDDP, not only when administered prior to CDDP, but also when administered at the same time as or subsequent to CDDP administration, without affecting the anticancer activity of CDDP. Thus, the administration of GSH is a promising approach for attenuating the nephrotoxicity caused by CDDP.