Background: Mesenchymal stem cells (MSCs) are pluripotent stromal cells that are among the most appealing candidates for regenerative medicine and may aid in the repair and regeneration of skeletal disorders through multiple mechanisms, including angiogenesis, differentiation, and response to inflammatory conditions. Tauroursodeoxycholic acid (TUDCA) has recently been used in various cell types as one of these drugs. The mechanism of osteogenic differentiation by TUDCA in hMSCs remains unknown. Methods: Cell proliferation was performed by the WST-1 method, and alkaline phosphatase activity and alizarin red-sulfate staining were used to confirm the osteogenic differentiation indicator. Expression of genes related to bone differentiation and specific genes related to signaling pathways was confirmed by quantitative real-time polymerase chain reaction. Results: We found that cell proliferation was higher as the concentration increased, and showed that the induction of osteogenic differentiation was significantly enhanced. We also show that osteogenic differentiation genes were upregulated, with the expression of the epidermal growth factor receptor (EGFR) and cAMP responsive element binding protein 1 (CREB1) being specifically high. To confirm the participation of the EGFR signaling pathway, the osteogenic differentiation index and expression of osteogenic differentiation genes were determined after using an EGFR inhibitor. As a result, EGFR expression was remarkably low, and that of CREB1, cyclin D1, and cyclin E1 was also significantly low. Conclusions: Therefore, we suggest that TUDCA-induced osteogenic differentiation of human MSCs is enhanced through the EGFR/p-Akt/CREB1 pathway.