Background <p>Endometriosis (EMs) is often classified as a benign disease; however, it exhibits malignant biological characteristics. Collapsin Response Mediator Protein 4 (CRMP4) has been implicated in the regulation of cell migration and invasion via its effects on the cytoskeleton, but its role in EMs remains unclear. This study investigates the role of CRMP4 in the pathogenesis of endometriosis, particularly in ectopic endometrial stromal cells (EcESCs).</p> Methods <p>We utilized a human endometrial ectopic stromal cell line (ihESCs), primary eutopic(Eu-ESCs), ectopic(Ec-ESCs) and control(CON-EuESCs) endometrial stromal cells, plus clinical samples. CRMP4 expression was disrupted with siRNA or lentiviral vectors; phenotypic changes were assessed by Transwell and wound-healing assays. A free-actin detection kit quantified polymerization and depolymerization. Stable ihESC cell lines overexpressing or knockout CRMP4 were analyzed by immunofluorescence, Western blot, and qPCR for MRTF/SRF axis activity after actin-modulating drugs. An in vivo mouse model received CRMP4-silencing lentivirus to evaluate lesion size and number; MRTF/SRF levels were examined by IHC, IF, WB and qRT-PCR.</p> Results <p>CRMP4 expression was significantly elevated in EcESCs and correlated positively with ASRM staging. CRMP4 promoted the polymerization of free actin, enhancing motility and invasiveness of endometrial stromal cells. It facilitated the nuclear translocation of MRTF, activating SRF and increasing the expression of downstream target genes related to migration and invasion. Targeting CRMP4 inhibited the growth of endometriosis lesions.</p> Conclusion <p>CRMP4 critically promotes actin polymerization and the invasive behaviors of ectopic mesenchymal cells in endometriosis by activating the MRTF/SRF axis. Targeting CRMP4 offers a promising therapeutic and diagnostic strategy for endometriosis.</p>