Cisplatin is an established chemotherapeutic drug for treatment of solid-organ cancers, and is the primary drug utilized in the treatment of head and neck cancer; however, cisplatin-induced nephrotoxicity largely limits its clinical use. Inhibition of sphingosine kinase 2 (SphK2) has been demonstrated to alleviate various kidney diseases. Therefore, we hypothesized that inhibition of SphK2 could also protect against cisplatin-induced nephrotoxicity. Results from the present study showed that the SphK2 inhibitor, ABC294640 or the knockdown of SphK2 by siRNA blocked the cisplatin-induced increase of cellular injury markers, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and Cleaved caspase-3 by Western blot analysis in HK-2 cells, a human renal tubular cell line. In addition, SphK2 inhibition blocked cisplatin-induced activation of NF-κB by Western blotting and Immunostaining analysis. Furthermore, SphK2 inhibition suppressed cisplatin-induced increases of proinflammatory markers, NLR Family Pyrin Domain Containing 3 (NLRP3), Interleukin-1β and Interleukin-6. Genetic deletion of the SphK2 gene in mice further confirmed that the inhibition of SphK2 protected against Cisplatin-induced kidney damage in vivo. Compared with wild type mice, SphK2 knockout mice exhibited less renal dysfunction and reduced promotion of kidney injury markers, inflammatory factors, tubular morphology damage, and fibrotic staining. At the same time, SphK2 inhibitor ABC294640 failed to interfere with the activity of cisplatin or radiation in two cell culture models of head and neck cancer. It is concluded that inhibition of Sphk2 protects against cisplatin-induced kidney injury. SphK2 may be used as a potential therapeutic target for the prevention or treatment of cisplatin-induced kidney injury.