Endometriosis is an estrogen-dependent neurovascular disorder characterised by growth of endometrial tissue (lesions) outside the uterine cavity. Patients suffer chronic pelvic pain and it has been proposed that co-recruitment of nerves/blood vessels (neuroangiogenesis) into the lesions is fundamental to the development of painful symptoms. We hypothesized that estrogen-dependent regulation of axonal guidance molecules of the SLIT/ROBO family could play a role in neuroangiogenesis occurring in endometriosis lesions found on the peritoneal wall. In tissue samples from human patients and a mouse model of endometriosis concentrations of mRNA encoded by SLIT3 were significantly higher in lesions than normal peritoneum. Estrogen regulation of SLIT3 was investigated using estradiol (E2) and selective agonists for each subtype of estrogen receptor (ERα agonist, PPT; ERβ agonist DPN). In mice, DPN (EC50 0.85) increased Slit3 mRNA concentrations compared to hormone depleted and E2-treated (EC50 0.1) animals and decreased the density of nerves but not vessels in endometriosis lesions. SLIT3 mRNA concentrations were increased in DPN-treated human endothelial cells (EC) and in PPT-treated (EC50:200) rat dorsal root ganglia neurons. Functional assays (neurite outgrowth, network formation) revealed that SLIT3 promotes angiogenesis but decreases neurogenesis. In conclusion, these data suggest estrogen-dependent expression of SLIT3 may play a key role in regulating nerve-vessel interactions within the complex microenvironment of endometriosis lesions.