In this study, we comprehensively profiled the ubiquitination landscape of endometriosis for the first time. Through proteomics, transcriptomics, and ubiquitylomics analyses of endometrial tissues from two patient cohorts, we uncovered significant dysregulation of ubiquitin-related enzymes and ubiquitinated proteins. By comparing tissues in pathological and healthy states, we delineated ubiquitinated proteins associated with the etiology of endometriosis and revealed their regulatory roles in relevant biological processes. We observed a pivotal role of ubiquitination in the fibrotic process, particularly in processes related to the activity of myofibroblasts and collagen deposition in ectopic tissues. Besides, we uncovered the downregulation of the E3 ubiquitin ligase TRIM33 in endometriotic tissues, along with its negative regulatory effect on fibrosis-related proteins TGFBR1/p-SMAD2/α-SMA/FN1 in vitro. It is important to note that, while our study provides compelling evidence supporting the crucial role of ubiquitination in endometriosis and the negative regulatory effect of TRIM33 on fibrosis, the specific regulatory mechanisms require further in-depth functional studies. This study contributes to a deeper understanding of endometriosis pathogenesis and highlights the potential of targeting ubiquitination pathways for therapeutic intervention in this debilitating condition.