Electrical and mechanical properties of myocardium vary transmurally in the left ventricular wall. Regional differences in the mechanical environment of cardiomyocytes may potentially contribute to this heterogeneity due to mechano-electric feedback. In the present study, we investigate transmural differences in active and passive tensions at different preloads between the subepicardial (EPI) and subendocardial (ENDO) cells isolated from mouse left ventricle, using our recently developed single cell stretch method where each cell end was held by a pair of carbon fibers to apply various extent of preload to the cells. To predict underlying mechanisms of the transmural differences, we used our electromechanical EPI and ENDO cell models to simulate experimentally obtained results. Wet experiments showed that both passive and active tensions at different preloads are higher in ENDO cardiomyocytes, indicating higher stiffness and contractility in ENDO cells compared to EPI cells. Our mathematical models reproduce experimental results, suggesting differences in the kinetics of cross bridges and calcium-troponin C complexes in ENDO and EPI models may essentially contribute to the differences in mechanical properties between the cells.