Pelvic organ cross-sensitization, also termed as viscero-visceral referred hyperalgesia, is a major contributor to painful endometriosis. Its underlying mechanism is poorly understood. Clinical and basic studies have shown that gabapentin, a drug that binds to the α2δ−1 subunit of voltage-dependent calcium channels (Cavα2δ−1), is effective in treating chronic visceral pain. Accordingly, we hypothesized that pelvic organ cross-sensitization in painful endometriosis is mediated by an upregulation of Cavα2δ−1 in the spinal cord. We examined if the dysregulation of spinal Cavα2δ−1 subunit may play an important role in the development of ectopic growths-to-colon cross-sensitization in a rat model of experimentally-induced endometriosis. Our findings suggest that there was an increased Cavα2δ−1 expression in the dorsal horn and an ectopic growths-to-colon cross-sensitization in female rats with established endometriosis. Intrathecal administration of gabapentin (300 μg) remarkably reduced the ectopic growths-to-colon cross-sensitization in rats with established endometriosis. Furthermore, intrathecal injection of Cavα2δ−1 antisense oligodeoxynucleotides reversed the ectopic growths-to-colon cross-sensitization and also normalized the upregulation of spinal Cavα2δ−1 expression in endometriosis rats. The current study suggests that the upregulation of Cavα2δ−1 in the spinal cord may contribute to pelvic organ cross-sensitization in painful endometriosis. Our study may provide a biological basis for selectively targeting this pathway to relieve viscero-visceral referred hyperalgesia in patients with painful endometriosis.