Abstract: The orientation of cellular alignment in smooth muscle tissue engineering is directly related to optimal movement of engineered tissue when it is transplanted in vivo. Cyclic mechanical strain has been applied to modulate the alignment, proliferation, and differentiation of smooth muscle cells. This study was conducted to investigate the effects of cyclic mechanical strain on primary cultured myofibroblasts seeded onto three-dimensional polymeric scaffolds, and to determine the optimal mechanical treatment time required to produce artificial smooth muscle. The cells were primary cultured from rabbit esophageal smooth muscle layer, and a self-designed stretching chamber was used to modulate the cells on porous polyurethane (PU) scaffolds with 10% strain at a frequency of 1 Hz. The applied cyclic strain induced cellular alignment. In particular, cellular alignment perpendicular to the direction of strain was generated in the condition strained over 18 h. In terms of proliferation, the strained groups differed significantly from the statically cultured group, but no difference was observed between groups that were subjected to straining for different lengths of time. Quantitative analysis of α-smooth muscle actin (SMA) showed that differentiation was significantly promoted at 18 h of strain. Penetration of primary cultured cells into the pores of PU scaffolds was shown after cyclic strain application, especially in 18 and 24 h of strain. Consequently, it is expected that myofibroblast/scaffold hybrids, cyclically strained in the defined time course, could be practically applied to organize functional smooth muscle tissues having consistent cell alignment and up-regulated SMA.