BACKGROUND:: Hedgehog signaling pathway is an important signal pathway, which regulates the development of variety of tissues and organs in the embryonic stage. It also plays an important role in osteogenesis. However, there are few data about the role of Hedgehog signaling molecules in osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. OBJECTIVE:: To investigate the expression of Hedgehog signaling molecules during osteogenic differentiation in rat BMSCs in vitro. METHODS:: BMSCs were derived from rat bone marrow, cultured and induced by dexamethasone. Identification of osteoblasts was performed by immunohistochemistry. Level of Sonic hedgehog (SHH) and Indian hedgehog (IHH) during osteogenic differentiation of BMSCs were detected by Western Blot. RESULTS AND CONCLUSION:: BMSCs were successfully obtained. Expression of collagen type I was increased on days 7, 14 and 21 following dexamethasone induction. In the osteogenic differentiation, a higher expression of SHH protein was detected in osteoblast induced group compared with non-induced group at the same time point (P < 0.05). While IHH expression was decreased and significantly lower in osteoblast induced group compared with non-induced group (P < 0.05). The results indicate that Hedgehog signaling molecules involved in the osteogenic differentiation of BMSCs induced by dexamethasone, and SHH and IHH had difference in osteogenic differentiation of BMSCs.