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Background Proteases and histamine, co-secreted by mast cells and bacteria, sensitise colonic nociceptors and contribute to irritable bowel syndrome (IBS) pain.Objective To determine whether irreversible proteolytic cleavage of protease-activated receptor-2 (PAR 2) and its continued activity in endosomes amplify and sustain otherwise transient pronociceptive actions of histamine receptors to cause recurrent pain, the defining symptom of IBS.Design We investigated PAR 2 and H1R coexpression in nociceptors using RNAscope and assessed the consequences of coactivation using electrophysiological assays of nociceptor sensitisation and biophysical measurements of receptor and effector activity.Results PAR 2 and H1R were co-expressed by human and mouse dorsal root ganglion nociceptors. Intracolonic infusion of faecal supernatants from patients with IBS with elevated histamine and proteolytic activity enhanced mechanosensitivity of colonic nociceptors in mice. Antagonists of PAR2 or H1R abolished this response. Combined administration of subthreshold concentrations of trypsin and histamine replicated the effects of faecal supernatant and caused hyperexcitability of isolated nociceptors. Pre-activation with trypsin sensitised histamine-induced hyperexcitability in nociceptors from wild-type but not Par2−/− mice. Endocytosis inhibitors prevented this hypersensitivity, consistent with sustained endosomal signalling of PAR2 and persistent nociceptor hyperexcitability. Trypsin amplified histamine-induced activation of H1R and β-arrestin2 and Gαq effectors at the plasmalemma and in endosomes. Conversely, histamine did not sensitise trypsin-induced hyperexcitability of neurons, in line with the inability of histamine to induce sustained nociceptor hypersensitivity.Conclusions By amplifying and maintaining the otherwise transient actions of H 1R and possibly other pain receptors, persistent PAR2 endosomal signalling makes a dominant contribution to IBS-related colonic pain.