Endometriosis (EM) is a benign gynaecological disease which shares some characteristics with malignancy like proliferation and invasion. So far, the pathogenesis of EM is still unclear. In this study, we investigated whether TRIM65 can play a role in the development of EM.TRIM65 expression levels in eutopic, ectopic and normal endometrium were detected by Quantitative real-time PCR and Western blot. Cell proliferation and invasion of primary endometrial stromal (EMS) cells were detected by CCK-8 and Transwell analysis. The interaction between TRIM65 and DUSP6 or C-myc was measured by co-Immunoprecipitation, ubiquitylation, dual luciferase and chromatin immunoprecipitation analysis.We found that TRIM65 was identified as an up-regulated gene in ectopic endometrial tissues and EMS cells compared with control groups without EM. TRIM65 expression was positively correlated with the levels of p-ERK1/2, C-myc, MMP-2 and Integrin β1 in ectopic endometrial tissues in patients and mice. TRIM65 promoted the cell proliferation and invasion of EMS cells via the ERK1/2/C-myc pathway through ubiquitination of DUSP6. C-myc promoted TRIM65 expression through inducing TRIM65 promoter activity. Additionally, the increased expression of TRIM65, C-myc, MMP-2, Integrin β1 and p-ERK1/2 and the decreased expression of DUSP6 in ectopic endometrial tissues were significantly suppressed by inhibition of ERK1/2 signaling pathway in ectopic endometrial tissues in experimental mice model.In conclusion, TRIM65 promotes invasion of ectopic EMS cells by activating a feedback loop with the ERK1/2/C-myc signaling pathway and may be a potential therapeutic target for EM.© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.