Endometriosis, an estrogen-dependent chronic inflammatory disease, is the most common cause of chronic pelvic pain (CPP). Here we investigated the effects of linaclotide, an FDA approved treatment for IBS-C, in a rat model of endometriosis. Eight weeks after endometrium transplantation into the intestinal mesentery, rats developed endometrial lesions as well as vaginal hyperalgesia to distension and decreased mechanical hindpaw withdrawal thresholds. Daily oral administration of linaclotide, a peripherally restricted guanylate cyclase-C (GC-C) agonist peptide acting locally within the gastrointestinal tract increased pain thresholds to vaginal distension and mechanical hindpaw withdrawal thresholds relative to vehicle treatment. Furthermore, using a cross-over design, administering linaclotide to rats previously administered vehicle resulted in increased hindpaw withdrawal thresholds, whilst replacing linaclotide with vehicle treatment decreased hindpaw withdrawal thresholds. Retrograde tracing of sensory afferent nerves from the ileum, colon and vagina revealed that central terminals of these afferents lie in close apposition to one another within the dorsal horn of the spinal cord. We also identified dichotomizing dual-labelled ileal/colon innervating afferents as well as colon/vaginal dual-labelled neurons and a rare population of triple traced ileal/colon/vaginal neurons within thoracolumbar DRG. These observations provide potential sources of cross-organ interaction at the level of the DRG and spinal cord. GC-C expression is absent in the vagina and endometrial cysts suggesting that the actions of linaclotide are via shared nerve pathways between these organs. In summary, linaclotide may offer a novel therapeutic option not only for treatment of chronic endometriosis-associated pain, but concurrent treatment of comorbid CPP syndromes.