Abstract Late-onset hypogonadism (LOH) refers to sexual and non-sexual symptoms in men caused by age-related decreases in circulating testosterone. Leydig cells (LCs) transplantation is considered to be one of a viable approach for LOH therapy, but the limited source of LCs limits the application of this approach. The aim of this study was to induce the directed differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into LCs in vitro, and explore the potential involvement of Wnt/β-catenin signaling pathway in the differentiation process. BMSCs were extracted from rats and characterized by flow cytometry for positive rates of mesenchymal stem cell markers CD29, CD44, CD90, and the hematopoietic marker CD45. BMSCs were divided into three groups: Control, Wnt agonist (CHIR-99021), and Wnt inhibitor (LGK-974), each incubated for 14 days. ELISA and RT-qPCR were used to verify the protein and mRNA expression of β-catenin, LRP5 and TCF, the key factors in Wnt/β-catenin signaling pathway. The average fluorescence intensity of 3β-hydroxysteroid dehydrogenase (3β-HSD) on the surface of LCs was detected by immunofluorescence (IF) assay. The content of testosterone secreted in cell culture medium was detected by ELISA. The results of flow cytometry indicated that we successfully extracted and cultured BMSCs. Moreover, post 14 days of incubation, the changes of β-catenin, LRP5 and TCF, at the protein and mRNA level demonstrate successful intervention in the activation and inhibition of the intracellular Wnt/β-catenin signaling pathway. Compared with the control group, the LCs surface marker 3β-HSD expression intensity in the CHIR-99,021 group was significantly increased by 69% (p