Objective To investigate the osteogenic differentiation of rabbit dental pulp stem cells (DPSCs) induced by transforming growth factor-β3 (TGF-β3) in vitro. Methods DPSCs and osteoblasts (OBs) were respectively obtained from rabbit dental pulp and skull by enzymetic digestion method. The morphology of the cells was observed by a light microscopy. Immunohistochemical staining and alizarin red staining were carried out to identify OBs. The third generation of DPSCs and OBs were divided into three groups, including DPSCs group (blank control), OBs group (positive control) and DPSCs+TGF-β3 group (experimental group). The expression of Runt-related transcription factor 2 (Runx-2) in each group was detected by immunohistochemical staining on the 5th day of culture. The activity of alkaline phosphatase (ALP) in each group was detected by assay kit on the 7th day of culture. Western Blot was used to detect the expression of the bone-specific markers Runx-2 and TGF-β3 proteins on the 1st, 3rd, 5th and 7th days of culture. Results The rabbit DPSCs were mostly long spindle-shaped with many synapses. The OBs were mostly short spindle-shaped or fibroblast-like, and plump with few synapses. The identification result showed that the DPSCs and OBs were positive. On the 5th day of culture, the expression of Runx-2 protein in the OBs group and DPSCs+TGF-β3 group showed strong positive. On the 7th day of culture, there was no significant difference in ALP activity between the above two groups (P>0.05). The results of Western Blot showed that the relative expressions of Runx-2 and TGF-β3 protein in the DPSCs group were significantly different from those in the other two groups, and the differences were statistically significant (all P<0.05). On the 7th day of culture, the relative expression of Runx-2 and TGF-β3 protein in the DPSCs+TGF-β3 group was higher than that in the other two groups, and the differences were statistically significant (all P<0.01). Conclusions TGF-β3 can promote the expression of early osteogenic specific proteins in DPSCs.