A sufficient cell source and minimal invasiveness in obtaining human adipose stromal cells (hASCs) hold great promise for their utilization in wound repair. However, little is known about how cell-residing microenvironments regulate the cellular response. In this study we explored the effects of polycaprolactone (PCL)/collagen nanofibers with distinct spatial arrangements (aligned and random) on phenotypic expression of hASCs in vitro. Elongated cell morphology, higher proliferation, and faster migration rate were observed for hASCs cultured on the aligned nanofibers, showing that hASCs could detect the nanofiber spatial arrangement and then distinctively respond. This study on the expression of extracellular matrix (ECM) related genes in hASCs revealed higher synthesis capacity for critical ECM molecules including tropoelastin, collagen I, and matrix metalloproteinase (MMP)-1 on the aligned nanofibers. Integrins α(5), β(1), β(3), β(6,) and transforming growth factor (TGF)-β(1) were differentially regulated by PCL/collagen nanofiber arrangements. Our results indicate that fiber orientation-induced phenotypic change of hASCs may be regulated by integrins and TGF-β signaling synergistically. These findings demonstrate the potential application of hASCs and aligned PCL/collagen nanofibers for accelerated wound repair.