To assess the use of heparin for the medical treatment of endometriosis-associated fibrosis.The effects of heparin on the endometriotic stromal cells (ECSCs)-mediated contractility were investigated.Research laboratory at a medical school.Endometriotic tissues from nine patients were used.Endometriotic stromal cells were cultured three dimensionally in the presence of heparin.The contractility of ECSCs was assessed by collagen gel contraction assay. Heparin-induced morphological changes of ECSCs were evaluated by laser scanning microscopy. The expression of contractility-related molecules in ECSCs was examined by Western blot analysis.In the presence of 10% fetal bovine serum, treated ECSCs showed significant collagen gel contractility (75.9% decrease in surface area after 48 hour vs. 0 hour controls). Endometriotic stromal cell-mediated gel contraction was significantly attenuated in the presence of heparin in a dose-dependent manner (55.7% reduction of the gel contraction at a concentration of 100 μg/mL of heparin sodium versus untreated controls after 48 hours). Heparin suppressed the ECSC attachment to collagen fibers. The expression of α-smooth muscle actin, Ras homology (Rho) A, Rho-associated coiled-coil-forming protein kinase (ROCK)-I, and ROCK-II was down-regulated by heparin administration.The present study suggests that heparin is a promising agent for the treatment of endometriosis-associated fibrosis. The inhibition of myofibroblastic differentiation, the attenuation of attachment to collagen fibers, and the suppression of Rho–ROCK-mediated pathway activation in ECSCs are involved in the action mechanisms of heparin.