Background: Chemotherapy resistance remains a barrier to survival of patients with head and neck squamous cell carcinoma (HNSCC).In this study, we investigated a potential mechanism ofmicroRNA-200s regulated glucosylceramide synthase (GCS) and GM3 synthase re-activating ceramide signaling pathway to overcome cisplatin-resistant HNSCC cell growth in vitro and in vivo.Methods: We established stably expressed miR-141,-200a,-429, and-200b HNSCC cell lines using a lentiviral delivering system.The levels of miR-200s and targeted proteins were analyzed by qRTPCR and western blot.The sensitizing drug resistance was evaluated by MTT, colony formation and flow cytometry, and tumor xenograft model.Results: We initially found a high expression level of key ceramide metabolic enzymes, glucosylceramide synthase (GCS) and GM3 synthase, coupled with a significant low level of eeramide in cisplatinresistant HNSCC cell lines compared to cisplatin-sensitive cell lines, such as GCS expression level was 9 folds higher in the resistant KBCP cells than the sensitive KB-3-1 cells.Further search indicates that GCS and GM3 synthase are two putative targets of the miR-200s.Thus, increased ceramide and decreased GCS and GM3 synthase were observed by re-expression of miR-200s.Furthermore, the miRNA-transfected cells and their tumor xenografts exhibited significantly slower growth than the mock-transfected cells and tumors, and regained their sensitivity to cisplatin.Conclusions: These results indicate that re-expression of the miR-200s could overcome the cisplatin resistance of HNSCC, at least in part by re-activating the ceramide signaling pathway, which provides a novel means to overcome the drug resistance problems.