Abstract: Objective： To explore the effects of SB431542, a TGF-β signaling pathway inhibitor, on the growing status and osteogenic differentiation of human gingival mesenchymal stem cells(hGMSCs) in vitro . Methods：The hGMSCs were isolated and cultured from clinically discarded gingival tissues. The surface markers of hGMSCs at passage 3 were by flow cytometry. Then the hGMSCs were treated with various concentrations of SB435142 (0, 0.1, 1, and 10 μmol/L) for the following detections. The proliferation and apoptosis of cells were analyzed using Cell Counting Kit-8 assay and Apoptosis Detection Kit, respectively. The osteogenic differentiation of hGMSCs was investigated using Alizarin Red staining after induced with osteogenic induction medium for 21 days. In addition, osteoblastic differentiation-related protein and downstream molecules of TGF-β signaling pathway in hGMSCs were measured by Western Blot analysis during osteogenic differentiation at the presence of SB431542(1μmol/L) or not. Results：The hGMSCs at passage 3 were positive for the typical MSC marker proteins CD105（99.8%）、CD90（100%）、and CD73（98.1%）,but negative for hematopoietic stem cell markers or macrophage and B-cells makers CD14（0.3%）、CD34（0.4%）、CD19（0.7%）、CD45（0.2%）、and HLA-DR（1.4%）. Apoptosis rates of the groups with SB431542 treatment (0.1,1, and 10μmol/L) were not statistically different with the control group（P＞0.05）.But the CCK-8 results showed that the proliferation of hGMSCs was inhibited in 10μmol/L SB431542 group, compared with control group at day 7(P < 0.5). After osteogenic induction for 21 days, 1μmol/L SB431542 treatment dramatically accelerated the calcified nodule formation compared to the control group（P < 0.5）.The collagen type I (COL-1), alkaline phosphatase（ALP）and runt-related transcription factor 2 (Runx2) protein expressions were remarkedly higher in SB431542 treatment group than that in control group after induced by osteogenic medium for 11 days（P < 0.5）. In addition, SB431542 treatment markedly inhibited the Smad3 phosphorylation in hGMSCs induced by osteogenic differentiation medium at 30 and 60 minutes（P < 0.5）. Conclusions：Treatment with 1μmol/L SB431542 could induce a robust osteogenic differentiation in hGMSCs in vitro, which may be regulated by inhibiting the phosphorylation level of Smad3 during osteogenic differentiation, indicating it is a great potential approach for repair and reconstruction of bone defect in future clinical application.