Background: Intrauterine adhesion (IUA) caused by endometrial mechanical injury has been found as a substantial risk factor for female infertility (e.g., induced abortion). Estrogen is a classic drug for the repair of endometrial injury, but its action mechanism in the clinical application of endometrial fibrosis is still unclear. Objective: To explore the specific action mechanism of estrogen treatment on IUA. Methods: The IUA model in vivo and the isolated endometrial stromal cells (ESCs) model in vitro were built. Then CCK8 assay, Real-Time PCR, Western Blot and Dual-Luciferase Reporter Gene assay were applied to determine the targeting action of estrogen on ESCs. Results: It was found that 17β-estradiol inhibited fibrosis of ESCs by down-regulating miR-21-5p level and activating PPARα signaling. Mechanistically, miR-21-5p significantly reduced the inhibitory effect of 17β-estradiol on fibrotic ESCs (ESCs-F) and its maker protein (e.g., α-SMA, collagen I, and fibronectin), where targeting to PPARα 3’-UTR and blocked its activation and transcription, thus lowering expressions of fatty acid oxidation (FAO) associated key enzyme, provoking fatty accumulation and reactive oxygen species (ROS) production, resulting in endometrial fibrosis. Nevertheless, the PPARα agonist caffeic acid counteracted the facilitation action of miR-21-5p on ESCs-F, which is consistent with the efficacy of estrogen intervention. result: We found that 17β-estradiol suppressed fibrosis of ESCs by downregulating miR-21-5p level and activating PPARα signaling. Mechanistically, miR-21-5p not only significantly suppress the inhibitory effect of 17β-estradiol on fibrotic ESCs (ESCs-F) and its maker proteins, including α-SMA, collagen I and fibronectin, but also target to PPARα 3’-UTR and prevent it being translated, thereby decreasing expressions of ACOX1 and CPT1A, the key enzymes in the downstream fatty acid oxidation (FAO) associated signaling pathway. Moreover, the PPARα agonist caffeic acid counteracted the facilitation action of miR-21-5p on ESCs-F. Conclusion: In brief, the above findings revealed that the miR-21-5p/PPARα signal axis played an important role in the fibrosis of endometrial mechanical injury and suggested that estrogen might be a promising agent for its progression. conclusion: It is revealed that miR-21-5p/PPARα signal axis played an important role in the fibrosis of endometrial mechanical injury and suggested that estrogen might be a promising agent for its progression.