BACKGROUND: Fibrogenesis is a common pathological feature of endometriotic lesions and contributes to the development of endometriosis-associated chronic pelvic pain and infertility. TGF-β is a critical factor in the induction of fibrogenesis; however, the underlying regulatory mechanisms of TGF-β-induced fibrosis in endometriosis remain unclear. In this study, we investigated the effects and mechanisms of CCN5 in regulating the progression of TGF-β-induced fibrosis in endometriosis. METHODS: We investigated the role of CCN5 in TGF-β-induced proliferation and pro-fibrotic responses in primary HESCs through CCN5 overexpression and knockdown techniques. We evaluated the impact of CCN5 modulation on the activation of the TGF-β/Smad and Wnt/β-catenin signaling pathways in primary HESCs subjected to TGF-β stimulation. To elucidate the role of Smad3 in CCN5 mediating TGF-β-induced pro-fibrotic response and the activation of TGF-β/Smad and Wnt/β-catenin signaling pathways, we employed SIS3, a specific inhibitor of Smad3. Additionally, we assessed the interaction between CCN5 and Smad3 in primary HESCs. RESULTS: The expression of CCN5 was significantly elevated at both the mRNA and protein levels in patients with endometriosis compared to healthy controls. Overexpression of CCN5 through transfection with LV-CCN5 notably attenuated TGF-β-induced proliferation and pro-fibrotic responses, whereas CCN5 knockdown exhibited the opposite effects in primary HESCs. Additionally, we observed the Wnt/β-catenin signaling pathway, a classical TGF-β-associated pro-fibrotic pathway, was significantly activated in primary HESCs under TGF-β stimulation. CCN5 overexpression inhibited the increased activity of both TGF-β/Smad and Wnt/β-catenin signaling pathways induced by TGF-β, while knockdown of CCN5 significantly enhanced TGF-β-induced activation of these pathways, an effect that was partially mitigated by the TGF-β inhibitor pirfenidone. An in vitro study demonstrated that SIS3, a specific Smad3 inhibitor, blocked the effects of CCN5 knockdown on TGF-β-induced proliferation and pro-fibrotic responses in endometriosis. Furthermore, we established that CCN5 directly interacts with Smad3 in cytoplasm, inhibiting Smad3's translocation into the nucleus and the subsequent activation of downstream target genes associated with TGF-β signaling pathways. CONCLUSIONS: CCN5 serves as an crucial negative regulator of fibrosis progression in endometriosis and represents a potential therapeutic target for endometrial fibrosis.