It has been widely recognized and proved that biophysical factors for mimicking in vivo conditions should be also considered to have stem cells differentiated into desired cell type in vitro along with biochemical factors. Biophysical factors include substrate and biomechanical conditions. This study focused on the effect of biomimetic mechanical stretching along with changes in substrate topography to influence on cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs). Elastic micropatterned substrates were made to mimic the geometric conditions surrounding cells in vivo. To mimic biomechanical conditions due to beating of the heart, mechanical stretching was applied parallel to the direction of the pattern (10% elongation, 0.5 Hz, 4 h/day). Suberoylanilide hydroxamic acid (SAHA) was used as a biochemical factor. The micropatterned substrate was found more effective in the alignment of cytoskeleton and cardiomyogenic differentiation compared with flat substrate. Significantly higher expression levels of related markers [GATA binding protein 4 (GATA4), troponin I, troponin T, natriuretic peptide A (NPPA)] were observed when mechanical stretching was engaged on micropatterned substrate. In addition, 4 days of mechanical stretching was associated with higher levels of expression than 2 days of stretching. These results indicate that simultaneous engagement of biomimetic environment such as substrate pattern and mechanical stimuli effectively promotes the cardiomyogenic differentiation of hMSCs in vitro. The suggested method which tried to mimic in vivo microenvironment would provide systematic investigation to control cardiomyogenic differentiation of hMSCs. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.