Background and Purpose: Bone marrow-derived mesenchymal stem cells (BMSCs) are potential in promoting bone regeneration for their multipotential differentiation capacity. Our previous study showed that carbon monoxide releasing molecule-3 (CORM-3) promoted the osteogenic differentiation of rat BMSCs. However, the mechanism was not clearly understood. MicroRNAs (miRNAs) play a critical role in regulating the osteogenic differentiation of BMSCs. We therefore investigated the role of miR-195-5p in CORM-3-induced osteogenic differentiation. Experimental Approach: The rat BMSCs were transfected with miR-195-5p mimics, miR-195-5p inhibitor, pcDNA3.1-Wnt3a, Wnt3a siRNA or their corresponding controls. The rat BMSCs osteogenic differentiation was assessed by quantitative real-time polymerase chain reaction, Western blot and alizarin red staining. In addition, dual luciferase assay was used for the verification of targeting relationship between miR-195-5p and Wnt3a. Key Results: miR-195-5p was down-regulated during the CORM-3-induced osteogenic differentiation of rat BMSCs. Overexpression of miR-195-5p inhibited CORM-3-induced osteogenic differentiation of rat BMSCs, evidenced by significantly decreased mRNA and protein expressions of runt-related transcription factor 2 and osteopontin, and matrix mineralization demonstrated. Whereas, inhibition of miR-195-5p expression enhanced osteogenic differentiation. miR-195-5p directly targeted Wnt3a. Overexpression of Wnt3a increased CORM-3-induced osteogenic differentiation of rat BMSCs, the opposite effect was observed in Wnt3a-deficient cells. Moreover, the inhibitory effect of miR-195-5p overexpression on CORM-3-induced osteogenic differentiation was rescued by Wnt3a overexpression. Conclusion and Implications: These results demonstrated that miR-195-5p may negatively regulate CORM-3-induced osteogenic differentiation of rat BMSCs by targeting Wnt3a, which provided insight into new mechanism of CORM-3, and theoretical basis for bone regeneration.