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BACKGROUND Recent studies have reported the tumor suppressive role of miR-5195-3p in the progression of several cancers, but the potential roles of miR-5195-3p in ovarian cancer (OC) remain largely unknown. METHODS We first analyzed the expression levels of miR-5195-3p in 83 pairs of human OC tissues and adjacent specimens by reverse transcription-quantitative PCR. The correlation of miR-5195-3p/rhomboid domain containing 1 (RHBDD1) and clinicopathological parameters was analyzed by chi-square test. The prognostic value of miR-5195-3p was evaluated by Kaplan-Meier method Cox proportional hazards models. The effects of miR-5195-3p on cell proliferation, cell cycle distribution, migration and invasion were examined by CCK-8 assay, colony formation assay, flow cytometry and transwell assay. Tumor forming was evaluated by nude mice model in vivo. The association between miR-5195-3p and RHBDD1 was verified by luciferase reporter assay. RESULTS We observed that miR-5195-3p level was remarkably reduced in OC tissues as compared to adjacent tissues. The expression of miR-5195-3p was associated with FIGO stage, depth of invasion and poor survival prognosis in OC patients. Overexpression of miR-5195-3p significantly suppressed cell proliferation, cell cycle G1/S transition, migration and invasion in OC cell lines (SKOV-3 and OVCAR3), while knockdown of miR-5195-3p obtained the opposite results. We further confirmed miR-5195-3p as a negative post-transcriptional modulator of RHBDD1. RHBDD1 expression was upregulated in OC tissues compared with adjacent tissues, which was inversely correlated with miR-5195-3p expression. The expression of RHBDD1 was associated with FIGO stage and distant metastasis. RHBDD1 overexpression reversed the suppressive role of miR-5195-3p on OC cell proliferation, migration and invasion. Consistent with the in vitro results, miR-5195-3p overexpression decreased the growth of subcutaneously inoculated tumors in nude mice. CONCLUSIONS Taken together, the present results indicated that miR-5195-3p acts a tumor suppressor by targeting RHBDD1 in OC.