AbstractTissue engineering and bone regeneration techniques using mesenchymal stem cells (MSCs) have started to be applied to the field of oral and maxillofacial surgery. Clinically, a shortened treatment time and improved efficiency are necessary because of the patients' needs and the running cost of cell culture. In the present study, the cultivation process for human MSCs (hMSCs) was examined by regulating the Wnt signaling pathway. We activated Wnt signaling with LiCl and inhibited Wnt signaling with sFRP‐3 (secreted Frizzled‐Related Protein‐3). The proliferation of LiCl‐treated hMSCs was examined by studying the cell growth rate and performing BrdU assays. Osteogenic differentiation of sFRP‐3‐treated hMSCs was examined by alizarin red staining, and osteogenic gene expression on days 7 and 14 after induction was examined by reverse‐transcription polymerase chain reaction (RT‐PCR) analysis and quantitative real‐time RT‐PCR analysis. LiCl‐treated hMSCs showed increased cell numbers and BrdU‐positive cells as compared to the untreated cells. Alizarin red staining showed early mineralization of hMSCs on day 7 of the sFRP‐3 treatment. A high expression level of the alkaline phosphatase gene on days 7 and 14 of sFRP‐3 treatment was also demonstrated. These results suggest that the regulation of the Wnt signaling pathway contributes to the increased cell numbers and the early osteogenic differentiation of hMSCs. This study supports the possibility that the regulation of the Wnt signaling pathway contributes to the development of effective and efficient bone regeneration techniques.