The process of osteogenic differentiation hinges upon the pivotal role of mechanical signals. Previous studies found that mechanical tensile strain of 2,500 microstrain (με) at a frequency of 0.5 Hz promoted osteogenesis in vitro. However, the mechanism of the mechanical strain influencing osteogenesis at the cellular and molecular levels are not yet fully understood. This study aimed to explore the mechanism of mechanical strain on osteogenic differentiation of MC3T3-E1 cells.