Background & Aims: Itch, like pain, is a major protective mechanism. Itch removes irritants from the skin, whilst pain allows withdrawal or avoidance of tissue damage. Painful and pruritogenic stimuli activate the peripheral endings of primary afferents that innervate second order neurons within the spinal cord. Whilst pain arises from both the skin and viscera; we investigated whether ‘itch-specific' pruritogenic mechanisms also have functions within visceral pathways. Methods: We assessed healthy mice and those with chronic visceral hypersensitivity (CVH) induced by TNBS. We also assessed mice over-expressing the bile acid receptor TGR5 (tgr5-tg) and mice lacking either TGR5 (tgr5 ) or TRPA1 (trpa1-/-). Protein and mRNA expression of the pruritogenic receptors TGR5, MrgprA3 (chloroquine receptor) and MrgprC11 (BAM8-22 receptor) in colonic afferent DRG neurons was assessed by immunofluorescence, single cell RT-PCR and quantitative RT-PCR. We determined colocalization of these receptors with the transient receptor potential (TRP) ankyrin 1 (A1) and vanilloid 1 (V1) ion channels and pruritogenic neuropeptides, gastrin-releasing peptide (GRP) and natriuretic poly-peptide B (NPPB). Using ex vivo electrophysiological recordings we determined the effects of TGR5 agonists (CCDC, DCA, oleanolic acid), as well as chloroquine and BAM8-22 on colonic afferent function. We also assessed the ability of these agonists to alter the transmission of colonic afferent signaling in vivo by measuring activation of dorsal horn neurons within the spinal cord. Results: Colonic DRG neurons express receptors for pruritogens, including TGR5 (15%), MrgprA3 (33%) and MrpgrC11 (33%), in addition to GRP (20%), NPPB (20%), TRPA1 (43%) and TRPV1 (64%). Notably, expression of TGR5, MrgprA3, and MrgprC11 was also evident in the colonic mucosal epithelium. In ex vivo and in vivo functional studies TGR5 agonists excite colonic afferents and amplify responses to mechanical stimuli, consistent with neuronal sensitization. These effects were greater in colonic afferents from tgr5-tg mice, and lost in afferents from tgr5-/and trpa1 mice. Chloroquine and BAM8-22 both excited colonic afferents and caused activation of dorsal horn neurons in the spinal cord. CVH mice showed a sustained amplification of the capacity of irritants to activate and sensitize colonic sensory neurons, suggesting functional up-regulation of this signalling pathway. Conclusions: Itch specific pruritogenic receptors are also expressed in colonic sensory pathways. In CVH mice colonic sensory pathways display increased responsiveness to known pruritogens. We propose that this colonic irritant sensing system is a visceral representation of the itch pathway in skin, and that it contributes to the sensory disturbances that accompany intestinal disorders, such as IBD and IBS. Supported by NHMRC Australia