Adipose-derived stem cells (ASCs) have been the subject of many tissue-engineering studies, mainly because of their multipotential properties. We have focused on the potential of human ASCs (hASCs) for smooth muscle cell (SMC) differentiation in cardiovascular tissue engineering. In this study, we investigated the combined effect of differentiation factors along with strain utilizing a customized device on hASCs proliferation and consequent differentiation into SMCs on an elastic nanofibrous scaffold. The cell proliferation increased in strain-stimulated culture and was more affected by media composition as compared to in static culture. Differentiation factors did not have an influence on SM α-actin (α-SMA) and myosin heavy chain (MHC) expression in static culture. However, α-SMA and MHC expression were affected by differentiation factors in strain culture, in particular, showing that treatment with retinoic acid significantly increased the expression of α-SMA (3.6-fold) and MHC (2-fold) as compared to strain alone. This study demonstrated the synergic effect of strain and biochemical factor on the SMC differentiation of hASCs and provided a useful method for the application of stem cells in mechano-active tissue engineering.