Spinal cord-mediated cross-organ sensitization between the uterus and the lower urinary tract underlies the high concurrence of pelvic pain syndrome and lower urinary tract dysfunctions, yet the role of gonadal steroids is still unknown. We tested the hypothesis that cross-organ sensitization on pelvic-urethra reflex activity caused by capsaicin instillation into the uterus is estrous cycle-dependent. When compared with a baseline reflex activity with single action potentials evoked by a test stimulation , capsaicin instillation into the uterine horn significantly increased reflex activity and this sensitization in reflex activity was corroborated by an increase in the expression level of the phosphorylated NR2B subunit. Intrauterine pretreatment with capsazepine, a TRPV1-selective antagonist, and an intrathecal injection of AP5, a NMDA antagonist, both abolished the cross-organ sensitization caused by the capsaicin instillation and the increment in the phosphorylated NR2B level. The amplitude of the cross-organ sensitization increased in a dose-dependent manner with the concentration of instilled capsaicin, from 100 to 300µM in both the proestrus and metestrus stages , whereas the amplitude weakened when the test concentrations were higher than 1000µM, indicating that desensitization can be induced using a high dose of capsaicin. Moreover, the cross-organ sensitization caused by the uterine capsaicin instillation increased significantly in the rats during the proestrus stage of the menstrual cycle when compared with the metestrus stage. These results suggest that estrogen levels might modulate the cross-organ sensitization between the uterus and the urethra and underlie the high concurrence of pelvic pain syndrome and lower urinary tract dysfunctions.