This study investigates the impact of sufentanil on endometrial cancer (EC) cells. Human EC cell lines (HEC-1A and Ishikawa) were exposed to different concentrations (10, 20 and 40 nM) of sufentanil for 48 hours, following which cell-counting-kit 8 (CCK8), clone formation and transwell assays as well as and western blot were conducted. The findings revealed that sufentanil reduced the cell viability, colony formation, migration and invasion of both HEC-1A and Ishikawa cells. Moreover, sufentanil treatment resulted in decreased levels of matrix metallo proteinase 9 (MMP-9), MMP-2, N-cadherin and alpha smooth muscle actin (α-SMA) proteins while increasing the expression of E-cadherin in treated cells. Furthermore, sufentanil treatment was associated with decreased phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt and mammalian target of rapamycin (mTOR), suggesting that its inhibitory effects on the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of EC cells could be attributed to the inactivation of the PI3K/Akt/mTOR pathway.