--- - |2 Chemoresistance remains a challenge in the effective treatment of solid tumors, including oral squamous cell carcinoma (OSCC). Mitochondrial dynamics and autophagy have recently been implicated in the chemoresistance of cancer cells. The neutralization of ceramide is also associated with multidrug resistance, and ceramide synthase 6 (CerS6) is known to induce apoptosis. However, whether CerS6 regulates chemoresistance in OSCC is not clearly understood. Therefore, we investigated the role of CerS6 in the susceptibility of OSCC cells to cisplatin. In this study, we observed that cisplatin‐resistant OSCC cells process lower levels of fission‐state mitochondria and cell apoptosis than cisplatin‐sensitive cells, and autophagy was activated in cisplatin‐resistant OSCC cells. We found lower CerS6 expression in cisplatin‐resistant OSCC cells. Overexpression of CerS6 with lentivirus‐encoded CerS6 complementary DNA in cisplatin‐resistant OSCC cells increased cisplatin sensitivity. Overexpression of CerS6 enhanced mitochondrial fission and apoptosis and attenuated cisplatin‐induced autophagy in cisplatin‐resistant OSCC cells. Further investigation indicated that CerS6 might function through altering calpain expression to enhance cisplatin sensitivity. Cisplatin‐resistant OSCC cells xenografted onto a nude mouse model confirmed that CerS6 enhanced cisplatin chemotherapy sensitivity to reduce tumor volume. These data indicate that CerS6 could mediate an effective response to cisplatin in chemoresistant OSCC. - Journal of Cellular Physiology, Volume 233, Issue 12, Page 9416-9425, December 2018.