Anticancer agent cisplatin may cause acute kidney injury (AKI) following renal inflammation and nephrotoxicity, which is characterized by tubular cell apoptosis and necrosis. In this study, we investigated the regulation and the role of cytokine macrophage migration inhibitory factor (MIF) in cisplatin-induced AKI. Immunohistochemistry showed that, in the kidney, MIF was mostly expressed in cortical tubular cells, and cisplatin caused MIF to be secreted by these cells in vivo and in vitro. MIF expression was not required for cisplatin-induced tubular cell apoptosis in vitro; pharmacologically inhibiting MIF attenuated cisplatin-induced AKI in vivo. Cellular MIF was secreted from cells after cisplatin-induced apoptosis, which was regulated by the epidermal growth factor receptor, Src, protein kinase C-δ, reactive oxygen species, mitogen-activated protein kinase, caspase, and DNA damage-activated ATR/ATM-regulated signaling pathways. We provide novel findings that cisplatin activates glycogen synthase kinase (GSK)-3 followed by GSK-3-mediated cell apoptosis, MIF secretion, and AKI. In a cisplatin-based chemotherapy model, inhibiting MIF prevented AKI without interfering with the anticancer efficacy; however, targeting GSK-3 was excluded as a potential treatment due to the GSK-3-dependent chemotherapeutic response. These results not only show that GSK-3 and MIF may facilitate cisplatin-induced AKI but also indicate a therapeutic strategy of inhibiting MIF to ameliorate AKI during cisplatin-based chemotherapy.