<strong>BACKGROUND</strong> Intrauterine adhesion (IUA) is a common reproductive system disease in women, characterized by endometrial stromal cell proliferation, increasing fibroblasts and increasing extracellular matrix secretion. The purpose of this study was to investigate the effect of mitomycin C on reducing endometrial fibrosis for IUA. <strong>MATERIAL AND METHODS</strong> Firstly, a rat IUA model was constructed by intrauterine mechanical injury. The endometrial stromal cells and fibroblasts were isolated and treated with mitomycin C. After that, Cell Counting Kit-8 (CCK-8) assay was used to investigate the endometrial stromal cell viability. Furthermore, cell cycle and apoptosis assays of endometrial stromal cells and fibroblasts were performed, respectively. Finally, the cell viability of human endometrial cells or human uterus adhesion fibroblasts treated with mitomycin C was determined using CCK-8 assay with or without estradiol. <strong>RESULTS</strong> Endometrial stromal cells were isolated from a rat IUA model. Cell cycle assay results showed that mitomycin C inhibited cell viability and promoted G1 cell cycle arrest and apoptosis in rat IUA endometrial stromal cells. Fibroblasts were also isolated from the rat IUA model. We found that mitomycin C inhibited the synthesis and secretion of collagen type I by western blotting analysis. Furthermore, mitomycin C promoted G1 cell cycle arrest and apoptosis in IUA rat uterine fibroblasts. We found that estradiol decreased the inhibitory effects of cell viability of human endometrial cells and human uterus adhesion fibroblasts by mitomycin C. <strong>CONCLUSIONS</strong> Our findings revealed that mitomycin C could reduce endometrial fibrosis for intrauterine adhesion.