Nonunion is a serious complication of a bone fracture that may occur in any bone of the skeletal system. It occurs when a broken bone fails to heal. Mesenchymal stem cell (MSC)-based tissue engineering technology has been considered an efficient method to improve the healing rate of nonunions. Although previous studies have demonstrated that bone morphogenetic protein 9 (BMP9) is highly capable of promoting the osteogenic differentiation of MSCs, the mechanisms involved remain largely unclear. In the present study, we investigated the possible involvement and the detailed role of Hedgehog (Hh) signaling in the BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 exerts an effect on Hh signaling in MSCs. The expression levels of early markers of BMP9-induced osteogenic differentiation, such as alkaline phosphatase (ALP) activity, and late markers of osteogenic differentiation, such as matrix mineralization, as well as the expression levels of osteopontin (OPN) and osteocalcin (OCN) were decreased by the Hh signaling inhibitor, cyclopamine, whereas these levels were increased by the Hh signaling agonist, purmorphamine. Furthermore, the BMP9-induced transcriptional activity of Smad1/5/8 and the expression of pivotal osteogenic transcription factors were reduced by cyclopamine, and were increased by purmorphamine. Taken together, our results demonstrate that BMP9 exerts an effect on Hh signaling in MSCs. What is most noteworthy, however, is that the inhibition or enhancement of Hh signaling resulted in the reduction and augmentation of the BMP9-induced osteogenic differentiation of MSCs, respectively, suggesting that Hh signaling is involved and plays a regulatory role in the osteogenic differentiation of MSCs induced by BMP9.