Objective <p>The aim of this study was to investigate the role and mechanism of Sulforaphane (SFN) in ameliorating endometriosis (EMs).</p> Methods <p>EMs’ rat model was established by autotransplantation method and administered with low, medium, and high doses of SFN. The volume and weight of the lesions were measured, the histopathological changes of the endometrium were observed by HE staining, and autophagy proteins were detected by immunofluorescence staining, whereas VEGF and apoptosis-related proteins were detected by Western blot. Rat primary endometrial stromal cells (ESCs) were isolated. Cell viability was determined by MTT, apoptosis by Tunel, migration and invasion by Transwell, and apoptotic and autophagic proteins by Western blot. Mitochondrial membrane potential, ATP content, and ROS levels were assessed for cellular mitochondrial function. Chloroquine (CQ), an autophagic flux inhibitor (AFI), was used alone or in combination with SFN to intervene cells.</p> Results <p>SFN inhibited the growth of EMs rat lesions in a dose-dependent pattern, alleviated the pathological changes of endometrium, downregulated VEGF, and upregulated apoptosis in endometrial tissues. Different concentrations of SFN effectively inhibited cell viability, migration, and invasion, and promoted apoptosis in rat ESCs, and the effects were concentration-dependent. SFN regulated autophagy proteins and inhibited autophagic flux. SFN disrupted the mitochondrial function of ESCs. CQ intervention alone inhibited ESCs cell viability, migration, and invasion, promoted apoptosis, and disrupted cellular mitochondrial function. Combined CQ intervention had a synergistic effect on SFN in regulating the biological phenotype and mitochondrial function of ESCs.</p> Conclusion <p>SFN ameliorates EMs in rats by modulating autophagy and mitochondrial dysfunction.</p>