The uterus plays an important and unique role during pregnancy and is a dynamic organ subjected to mechanical stimuli. It has been reported that infertility occurs when the peristalsis is prevented, although its mechanisms remain unknown. In this study, we found that mechanical strain mimicking the peristaltic motion of the uterine smooth muscle layer enabled the endometrial stromal cells to acquire contractility. In order to mimic the peristalsis induced by uterine smooth muscle cells, cyclic tensile stretch was applied to human endometrial stromal cells. The results showed that the strained cells exerted greater contractility in three-dimensional collagen gels in the presence of oxytocin, due to up-regulated alpha-smooth muscle actin expression via the cAMP signaling pathway. These in vitro findings underscore the plasticity of the endometrial stromal cell phenotype and suggest the possibility of acquired contractility by these cells in vivo and its potential contribution to uterine contractile activity. This phenomenon may be a typical example of how a tissue passively acquires new contractile functions under mechanical stimulation from a neighboring tissue, enabling it to support the adjacent tissue's functions.