Background: Hyaline cartilage defects exhibit a major challenge in the field of orthopedic surgery owing to its limited repair capacity. On the other hand, mesenchymal stem cells (MSCs) are regarded as potent cells with a property of cartilage regeneration. We aimed to optimize marrow-derived MSC chondrogenic culture using a small bioactive molecule referred to as BIO. Methods: MSCs from the marrow of NMRI mice were extracted, culture-expanded, and characterized. Micro-mass culture was then established for chondrogenic differentiation (control group). The cultures of MSC in chondrogenic medium supplemented with 0.01, 0.05, 0.1, and 1 µM BIO were taken as the experimental groups. Cartilage differentiation was examined by both histological sections and real-time PCR for Sox9, aggrecan, and collagen II at different time points. Moreover, the involvement of the Wnt pathway was investigated. Results: Based on histological sections, there was seemingly more intense metachromatic matrix produced in the cultures with 0.01 µM BIO. In this experimental group, cartilage-specific genes tended to be upregulated at day 14 compared to day 21 of the control group, indicating the accelerating effect of BIO on cartilage differentiation. Overall, there was statistically a significant increase (P=0.01) in the expression level of cartilage-specific genes in cultures with 0.01 µM BIO (enhancing effects). These upregulations appeared to be mediated through the Wnt pathway evident from the significant upregulation of T-cell factor and beta-catenin molecules (P=0.01). Conclusion: Taken together, BIO at 0.01 µM could accelerate and enhance in vitro chondrogenesis of mouse marrow-derived MSCs.