Objective To explore the influence of miR-486-5p on the malignant behavior of endometrial cancer(EC) cells and its targeted regulation of PTEN. Methods The expression miR-486-5p and PTEN mRNA in EC tissues and cells was detected by qRT-PCR. The correlation between miR-486-5p and PTEN mRNA expression in EC tissues and the clinical characteristics of patients were analyzed. A dual luciferase reporter assay was performed to verify the relationship between miR-486-5p and PTEN. Ishikawa cells were separated into NC, NC inhibitor, miR-486-5p inhibitor, miR-486-5p inhibitor+si-NC, and miR-486-5p inhibitor+si-PTEN groups. Western blot was performed to detect expression of PTEN and PI3K/ AKT pathway-related proteins in cells. CCK-8 assays were performed to assess cell viability. Colony formation assays were used to examine the ability of cells to form colonies. Flow cytometry was performed to assess apoptosis, Scratch healing and transwell assays were applied to assess cell migration and invasion. Results The miR-486-5p was highly expressed in EC tissues and cells, while PTEN mRNA was expressed at a low level (P<0. 05). miR-486-5p and PTEN mRNA expression in EC tissues was related to the tumor size, differentiation degree, International Federation of Gynecology and Obstetrics stage, and lymph node metastasis (P<0. 05). miR-486-5p in Ishikawa cells might negatively target and regulate PTEN expression. Compared with NC and NC inhibitor groups, cell viability at 48 and 72 h, scratch healing rate, and ratios of p-PI3K/ PI3K and p-AKT/ AKT of Ishikawa cells in the miR-486-5p inhibitor group were decreased (P<0. 05), the number of colonies formed and the numbers of migrating and invasive cells were decreased (P<0. 05), and the apoptosis rate was increased (P<0. 05). Compared with miR-486-5p inhibitor and miR-486-5p inhibitor+si-NC groups, cell viability at 48 and 72 h, scratch healing rate, and ratios of p-PI3K/ PI3K and p-AKT/ AKT of Ishikawa cells in the miR-486-5p inhibitor+si-PTEN group were increased (P< 0. 05), the number of colonies formed and the numbers of migrating and invasive cells were increased (P<0. 05), and the apoptosis rate was decreased (P<0. 05). Conclusions Inhibition of miR-486-5p may suppress activation of the PI3K/ AKT pathway by negatively targeting PTEN, thereby inhibiting the proliferation, migration, and invasion of EC cells and inducing their apoptosis.