Background Accumulating evidence indicates that miR-5195-3p exerts tumor-suppressive roles in several tumors. However, the clinical significance and biological function of miR-5195-3p in prostate cancer (PCa) have not been reported yet. Methods The expression levels of miR-5195-3p and Cyclin L1 (CCNL1) were determined using quantitative real-time PCR in clinical specimens and cell lines. The clinical significance of miR-5195-3p in patients with PCa was evaluated using Kaplan-Meier survival analysis and Cox regression models. Cell proliferation and cell cycle distribution were measured by CCK-8 assay and flow cytometry, respectively. The association between miR-5195-3p and CCNL1 was analyzed by luciferase reporter assay. Results MiR-5195-3p expression levels were significantly downregulated in 69 paired PCa tissues compared with matched adjacent normal tissues. The decreased miR-5195-3p expression was associated with Gleason score and TNM stage, as well as worse survival prognosis. The in vitro experiments showed that miR-5195-3p overexpression suppressed the proliferation and cell cycle G1/S transition in PC-3 and DU145 cells. Elevated miR-5195-3p abundance obviously impaired tumor formation in vivo using PC-3 xenografts. Mechanistically, CCNL1 was a direct target of miR-5195-3p in PCa cells, which was inversely correlated with miR-5195-3p in PCa tissues. Importantly, CCNL1 knockdown imitated, while overexpression reversed, the effects of miR-5195-3p overexpression on PCa cell proliferation and cell cycle G1/S transition. Conclusions Our data suggest that miR-5195-3p functions as a tumor suppressor by targeting CCNL1 in PCa.