Background: Endometriosis is a benign gynecological disorder which shares certain characteristics with malignant tumor like migration, invasion and proliferation. Glioma-associated oncogene homolog 1 (GLI1) has been implicated in some cancers including endometrial cancer, however, its role in endometriosis remains unknown. Methods: The aim of this study was to explore the expression pattern of GLI1 in endometriosis, and further investigate the effect of GLI1 regulation on human endometrial stromal cells. The expression of GLI1 in normal endometrium and ectopic tissues was analyzed by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The Short hairpin RNA (ShRNA) intervention technique and GLI1 inhibitor GANT-61 were used to silence GLI1. The expression levels of GLI1, MMP2 and MMP9 was detected by qRT-PCR and western blot. The migration and invasion ability of human endometrial stromal cells was determined by wound healing assay and transwell migration/invasion assay. The viability and proliferation potentiality of cells was detected by MTT assays and colony formation assay, respectively. Results: We found that the expression of GLI1 mRNA and protein were significantly higher in ectopic endometrium from patients with endometriosis. Our analyses also show that GLI1 downregulation attenuated cells migration, invasion and proliferation abilities. What's more, reduced expression of GLI1 inhibited the expression of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9). Conclusions: Our findings suggest that high levels of GLI1 may contribute to the development of endometriosis by promoting cell migration, invasion and proliferation involving regulation of MMP2 and MMP9 expression. Therefore, inhibition of GLI1 might be a novel potential therapeutic approach to the treatment of endometriosis, which sheds new light on our understanding of the pathogenesis of endometriosis.