MicroRNA-365 (miR-365) has been reported to play an important role in tumorigenesis in many types of cancers; however, the role of miR-365 in the carcinogenesis of ovarian cancer remains unknown. In this study, we focused on the roles and underlying mechanisms of miR-365 in ovarian cancer. Here, we found that miR-365 expression level was significantly decreased in ovarian cancer tissues and cell lines, and that low miR-365 expression was negatively significantly associated with advanced stages as defined by the International Federation of Gynecology and Obstetrics (FIGO), histological grading, and lymph node metastasis. Further functional assays showed that transfection with a miR-365 mimic significantly decreased ovarian cancer cell proliferation, colony formation, migration, and invasion. In addition, Wnt5a was identified as a target gene of miR-365 in ovarian cancer by bioinformatic analysis, luciferase reporter assay, qPCR, and western blot. Wnt5a expression levels were upregulated and inversely correlated with miR-365 expression in ovarian cancer tissues (r = -0.638, P < 0.0001). Overexpression of Wnt5a could effectively reverse the miR-365 overexpression-induced suppression of proliferation and invasion in ovarian cancer cells. Additionally, in vivo studies utilizing a xenograft model demonstrated that overexpression of miR-365 could reduce tumor growth by repressing Wnt5a. Taken together, these findings suggest that miR-365 may be a promising candidate for therapeutic application in ovarian cancer treatment.