We aimed to explore the mechanisms of sunitinib-resistance in renal cell cancer (RCC) and provide new therapeutic evidence and biomarkers for RCC treatment using human clear-cell renal cell carcinoma (ccRCC) cell line, 786-O. Microarray analysis, quantitative real time PCR (qRT-PCR), Western blot, and immunohistochemistry were used to detect Ecto-5 '-nucleotidase (NT5E) expressions in sunitinib-resistant tissues in RCC. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, Thiazolyl Blue Tetrazolium Bromide (MTT), cell-count kit 8 (CCK-8), wound healing, and Transwell assays were used to investigate the influences of sunitinib and NT5E on 786-O cell line and sunitinib-resistant 786-O cell line (S786-O). Protein expressions related to epithelial-mesenchymal transition (EMT) and AKT/GSK-3 beta signaling pathway in 786-O cells and S786-O cells were determined by Western blot. In vivo experiment was performed using NCr-nu/nu mice to explore the effects of NT5E on cell growth and EMT signal in sunitinib environment. NT5E expression was upregulated in sunitinib-resistant RCC tissues and cells. NT5E downregulation repressed RCC cell proliferation, migration as well as invasion. EMT course and AKT/GSK-3 beta signal pathway both in vitro and in vivo in sunitinib environment was suppressed to varying degrees via NT5E inhibition. NT5E inhibition could suppress the growth of sunitinib-resistant RCC cells and restrain EMT course and AKT/GSK-3 beta signal pathway in sunitinib environment in RCC. (c) 2018 IUBMB Life, 71(1):113-124, 2019