Osterix is an essential transcription factor for osteogenesis and is expressed in osteoblasts. Although Osterix has been shown to be induced by bone morphogenetic protein 4, the molecular mechanism underlying Osterix function during osteoblast differentiation remains unclear. Connexin43 (Cx43) is the most abundant gap junction protein in bone cells and plays a critical role in osteoblast differentiation. However, little is known about the functional interactions between Osterix and the Cx43 promoter. In the present study, we investigated the relationship between Osterix and Cx43 in HEK293 and C2C12 cells. Cx43 expression was significantly repressed by the addition of shRNA against Osterix, whereas overexpression of Osterix resulted in enhanced Cx43 expression. Furthermore, Osterix directly occupied the promoter region of Cx43 and subsequently increased Cx43 promoter activity in a dose-dependent manner. In addition, phosphorylation of the Ser76 and Ser80 residues in Osterix were found to be critical for its activity on the Cx43 promoter. Our results suggest that Osterix plays an important role in increasing bone morphogenetic protein 4-induced Cx43 activity.