The object of this study is to investigate the effects of intermittent cyclic stretching on the smooth muscle cells (SMCs) seeded onto aligned multi-layered fibrous scaffold. To make multi-layered fibrous scaffold, polyurethane (PU) and poly(ethylene oxide) (PEO) were electrospun alternatively, then were immersed into distilled water to extract PEO. Various types of scaffolds were fabricated depending on fiber directions, i.e., aligned or randomly oriented. The direction of stretching was either parallel or vertical to the fiber direction for the aligned scaffolds. The stretching was also applied to the randomly aligned scaffolds. The duration of stretching was 2 min with 15 min resting period. During the stretching, the maximum and minimum strain was adjusted to be 10 and 7%, respectively with the frequency of 1 Hz. The bioactivities of cells on the scaffolds were assessed by quantifying DNA, collagen, and glycosaminoglycan (GAG) levels. And the cell morphology was observed by staining F-actin. SMCs under parallel stretching to the fiber direction responded more positively than those in other conditions. From the results, we could explain the morphological effect of a substrate on cellular activities. In addition the synergistic effects of substrate and mechanical stimuli effects were confirmed.