Mechanical forces are widely involved in regulating basic cellular functions, including proliferation, differentiation, adhesion, and migration. However, few studies show how the mechanical forces impact cell division direction, a crucial process in development, differentiation, embryogenesis and recovery of tissues. It has been documented that cell division direction is guided by stress fibers, which are cortical cues perpendicular to exogenous cyclic uniaxial stretch. In our study, we used the murine 3T3 fibroblasts as a model, to investigate the effect of uniaxial stretch on stress fiber alignment and cell division direction with custom-made stretch devices. We found that cyclic uniaxial stretch induced stress fiber alignment and cell division direction perpendicularly to the stretch direction. The blockage of actin assembly and myosinⅡ, two basic components of stress fibers, resulted in a disoriented cell division under uniaxial stretch, which suggested that the cell division direction was secondary to the stress fiber alignment. Our data suggested that Rho/Rho-kinase/MLC (Myosin Light Chain) and MLCK/MLC pathways were involved in the process of stretch-induced stress fiber alignment and cell division direction. Taken together, the present work demonstrated an important effect of uniaxial stretch on cell division direction via affecting the stress fibers alignment.