Bromodomain 4 (BRD4), an important epigenetic regulator, is involved in many bone-related pathologies via promoting osteoclast formation. However, whether and how it participates in the process of osteoblast formation remain unclear. This study aimed to investigate the potential role of BRD4 in osteogenic differentiation of bone marrow stromal cells (BMSCs). Our experiments revealed that an inhibitor of BRD4, JQ1, attenuated osteogenic differentiation of BMSCs. The recombinant adenoviruses for AdBRD4 and AdsiBRD4 could infect BMSCs with high efficiency. Exogenous BRD4 expression potentiated differentiation, and silencing endogenous BRD4 expression decreased it. In addition, the Wnt/β-catenin signaling pathway is known to be important for osteogenic differentiation. Our results showed that AdBRD4 increased the expressions of Wnt3a and β-catenin while AdsiBRD4 decreased the expressions. What's more, the recombinant adenovirus for Adsiβ-catenin, which obviously decreased in β-catenin expression, inhibited BRD4-induced osteogenic differentiation. Conclusion: Our data indicates that the epigenetic reader BRD4 participates in the process of BMSC osteogenic differentiation via the Wnt/β-catenin signaling pathway. This finding may pave the way into further understanding the mechanism of BMSC osteogenic differentiation.