Cisplatin is a highly successful chemotherapeutic agent used for the treatment of solid tumors. However, nephrotoxicity is a limiting factor that occurs in 30% of patients under treatment. Many mechanisms are involved in cisplatin-induced nephrotoxicity, such as epithelial and endothelial injury, inflammation, oxidative stress, and renal vasoconstriction. The mineralocorticoid receptor (MR) has an important role in inflammation and vascular function. MR blockage and ablation have been shown to be effective in preventing renal ischemia-reperfusion injury and cyclosporine A-induced nephrotoxicity. We investigated whether MR antagonism with spironolactone or eplerenone could prevent cisplatin-induced nephrotoxicity. Here, we show that spironolactone treatment exacerbates nephrotoxicity in mice treated with acute and long-term cisplatin regimes. Moreover, spironolactone potentiated the toxicity induced by cisplatin treatment in a cell viability assay in human embryonic kidney cells. In contrast, eplerenone neither prevented nor increased cisplatin toxicity in mice or cultured cells. Thus, our studies support recent findings showing that spironolactone potentiates cisplatin-induced cytotoxicity, independently of mineralocorticoid receptor inhibition.