Mechanical stimuli are responsible for bone remodeling during orthodontic tooth movement. The role of mechanical stimulation in the regulation of the fate of bone mesenchymal stem cells (BMSCs) is of interest in bone regeneration and tissue engineering applications. However, the signaling pathway involved in strain-induced biochemical events in BMSCs is not well established and can be controversial. This study investigated strain-induced proliferation and differentiation of BMSCs, as well as the mechanism of mechanotransduction. BMSCs were exposed to continuous mechanical strain (CMS) of 10% at 1 Hz. The results showed that CMS reduced the proliferation of BMSCs and stimulated osteogenic differentiation by activating Runx2, followed by increased alkaline phosphatase (ALP) activity and mRNA expression of osteogenesis-related genes (ALP, collagen type I and osteocalcin). Furthermore, the phosphorylation level of extracellular regulated protein kinase (ERK)1/2 increased significantly at the onset of strain. However, the presence of U0126, a selective inhibitor of ERK1/2, blocked the induction of Runx2 and subsequent osteogenic events. These findings demonstrate that CMS regulated Runx2 activation and favored osteoblast differentiation through activation of the ERK1/2 signaling pathway. These results will contribute to a better understanding of strain-induced bone remodeling and will form the basis for the correct choice of applied force in orthodontic treatment.