The osteogenic capacity of mesenchymal stem cells (MSCs) and the importance of β-adrenergic signals in bone formation and resorption have been well investigated. However, little is known about the development of β-adrenergic receptor (β-AR) systems and the role of β-adrenergic signals in osteogenic differentiation of MSCs, which is critically important in bone physiology and pharmacology. In this study, we demonstrated that both the mRNA and protein levels of β2- and β3-AR are up-regulated following osteogenesis of mouse MSCs. We also established that β-AR agonists negatively while antagonists positively affect MSC osteogenesis. Both β2- and β3-AR are involved in MSC osteogenesis, with β2-AR being dominant. The effect of β-ARs on MSC osteogenesis is partly mediated via the cAMP/PKA signaling. These findings suggest that MSC is also a target for β-adrenergic regulation and β-adrenergic signaling plays a role in MSC osteogenesis.