Endometriosis (ENDO) is a disease common in women that is defined by abnormal extra uteral growths of uterine endometrial tissue and associated with severe pain. Recent studies in a rat model and women showed that sensory and sympathetic nerve fibers sprout branches to innervate the abnormal growths. Furthermore, the endocannabinoid system is involved in both endometriosis and its associated pain. It is not clear whether cannabinoid receptor 1 (CB1R) is involved in the sensitivity of afferent nerve innervating ectopic endometrium to capsaicin (Cap)/bradykinin (BK) Herein, using an ex vivo ectopic endometrium preparation from rat model of endometriosis, we examined the sensitivity of afferent nerve innervating ectopic endometium to Cap/BK, and the effects of CB1R agonist ACPA on Cap/BK-evoked activity of afferent nerve. ENDO model: female Sprague-Dawley rats (200-225g, n=9) in diestrus were anesthetized with sodium pentobarbital (50 mg/Kg, i.p.), four pieces of uterine horn (2×2 mm) were sewn around alternate cascade mesenteric arteries starting from the caecum, which will form ectopic uterine cysts. Recording of afferent nerve innervating ectopic cyst was performed in an ex vivo preparation 10 weeks after ENDO. The preparation was superfused continuously at a rate of approximately 15 ml/min with oxygenated (95%O2/5%CO2) Krebs solution at 37 °C. ACPA (300 nM) diluted in Krebs superfused the preparation 10 min before either Cap (300 nM) or BK (300 nM) superfusion. Spike 2 software was used to calculate the change of afferent activity. Values are means ± S.E.M., compared by ANOVA. The response of afferent nerve innervating ectopic cyst either to Cap or BK was changed compared to that in ex vivo naïve ileum preparation. The peak increase of afferent activity was occurred at 90 sec after Cap in ex vivo either ectopic cyst or naïve ileum preparation, while the Cap-induced afferent activity was decreased followed by increase in ex vivo ectopic cyst preparation. The BK-induced increase in afferent activity was occurred at 90 sec and 3 min after BK in ex vivo naïve ileum and ectopic cyst preparations, respectively. The peak increase in afferent activity induced by either Cap or BK was inhibited by ACPA (8.33 ± 0.89 vs. 4.35 ± 0.36 imp/30s, Cap alone vs. Cap + ACPA, p < 0.05; 12.37 ± 0.85 vs. 7.33 ± 0.34 imp/30s, BK alone vs. BK + ACPA, p < 0.05). However, ACPA did not affect the response of afferent nerve to either Cap or BK in ex vivo naïve ileum preparation. These findings indicate that peripheral CB1R affects the sensitivity of afferent nerve innervating ectopic endometrium to capsaicin or bradykinin.