Abstract: Background Columbin, a diterpenoid furanolactone-type compound isolated from the traditional Chinese herb Tinospora sagittate, exhibits diverse biological activities, and the effects of columbin on the osteogenic differentiation of bone marrow mesenchymal stem cells remain elusive.Objective To study the effect of columbin on osteogenic differentiation of human jaw bone marrow mesenchymal stem cells and explore its molecular mechanism. Methods The extracted h-JBMMSCs were identified by flow cytometry and osteogenic and lipogenic differentiation; h-JBMMSCs were subjected to osteogenic induction in the presence of columbin at concentrations of 0, 5, 10, 20, 40, and 80 μmol/L. The effect of columbin on cell proliferation was detected by CCK-8 assay; ALP activity assay, alizarin red staining and calcium ion semi-quantitative assay were used to evaluate the effect of columbin on osteogenic differentiation of h-JBMMSCs; The expression of Runx2, Col1a1 proteins and mRNA were detected by Western blot and q-PCR; Bioinformatic analysis of transcriptome data and in vitro experiments were conducted to investigate the molecular mechanism of columbin in promoting osteogenic differentiation of h-JBMMSCs. Results Columbin exhibited no inhibitory effect on the proliferation capacity of h-JBMMSCs at concentrations ≤20 µmol/L (P<0.05). Compared with the 0 µmol/L group, the ALP activity was increased in the 5 µmol/L group (P<0.01). The alizarin red staining area and the density of calcium nodules increased in 20 µmol/L columbin group (P＜0.001).The expressions of osteogenesis-related genes and proteins Runx2 and Col1a1 were increased after the osteogenic induction of columbin (P＜0.05).The results of enrichment analysis showed that differentially expressed genes were significantly enriched in ECM receptor interaction and P53 signaling pathway, further analysis of box plots and in vitro experiments showed that the mRNA and protein expression of β-catenin, the main downstream effector of Wnt signaling pathway, significantly increased after columbin-induced osteogenesis. Conclusion Columbin at concentrations ≤20 µmol/L exerted a promotive effect on the osteogenic differentiation of h-JBMMSCs, and this process may involve the activation of the Wnt signaling pathway.