Objective To investigate the effects of Yunnan Baiyao on promoting osteoblast proliferation and differentiation, and to provide theoretical basis for its bone repair mechanism. Methods Cranium was harvested from fetal SD rats. Then, osteoblast was acquired and cultured by repeating enzymatic digestion-adherent explant method identified via invert microscope, alkaline phosphorase stain, and stain of calcified nodules. After using different concentrations of Yunnan Baiyao stimulating osteoblasts, the proliferation of the acquired cells was examined through methyl thiazolyl tetrazolium assay. Alkaline phosphatase(ALP) activity and osteocalcin content were analyzed by using a spectrophotometer and enzyme linked immunosorbent assay in the cell lysate liquid. Results Compared with the controls, three different concentration groups can promote the proliferation of osteoblast; the 5 mg·L-1group had more obvious roles in 5 and 7 d. It also increased the activity of ALP and promoted osteocalcin synthesis simultaneously. Conclusion Yunnan Baiyao can promote proliferation and differentiation of osteoblasts.