Context: Decidualization of the endometrium involves the morphological and biochemical reprogramming of the estrogen-primed proliferative endometrial stromal compartment under the continuing influence of progesterone., Objectives: The aim of this study was to evaluate the involvement of the extracellular matrix contractility of eutopic and ectopic endometrial stromal cells during the tissue remodeling processes associated with decidualization., Design: The effect of decidualization on the contractile profile of the endometriotic cyst stromal cells and eutopic endometrial stromal cells with or without endometriosis in the three-dimensional collagen gel culture was investigated using laser scanning microscopy, collagen gel contraction assays, and Western blot analysis., Results: Decidualized ectopic and eutopic endometrial stromal cells in the three-dimensional collagen gel culture mimicked the morphology of decidual tissue in vivo. In vitro decidualization inhibited the contractility of these eutopic and ectopic endometrial stromal cells. Down-regulation of integrin alpha1beta1 and alpha2beta1 expression, suppression of Ras homology A (Rho A), Rho-associated coiled-coil-forming protein kinase (ROCK)-I and ROCK-II expression, inhibition of the differentiation into the myofibroblastic phenotype, and induction of differentiation into epithelioid decidual phenotype were observed in these cells during decidualization., Conclusions: It is suggested that the attenuation of eutopic endometrial stromal cell-mediated contractility by decidualization is a novel and integral mechanism of the physiological endometrial tissue remodeling process during menstrual cycles. Although ectopic endometrial stromal cells have enhanced contractile profile, decidualization can attenuate the contractility of these cells. These findings may be one of the action mechanisms by which oral contraceptives and progestins ameliorate endometriosis.