Opioid tolerance (OT) leads to dose escalation and serious side effects, including opioid-induced hyperalgesia (OIH). We sought to better understand the mechanisms underlying this event in the gastrointestinal tract. Chronic administration of morphine by intraperitoneal (i.p.) injection in male C57BL/6 mice evoked tolerance and evidence of OIH in an assay of colonic afferent nerve mechanosensitivity; this was inhibited by the δ-opioid receptor (DOPr) antagonist naltrindole when i.p. injected previous morphine administration. Patch clamp studies of dorsal root ganglia (DRG) neurons following overnight incubation with high concentrations of morphine, the µ-opioid receptors (MOPr) agonist DAMGO or the DOPr agonist DADLE evoked hyperexcitability. The pronociceptive actions of these opioids were blocked by the DOPr antagonist SDM25N but not the MOPr antagonist CTOP. The hyperexcitability induced by DAMGO was reversed after a 1 hr washout but reapplication of low concentrations of DAMGO or DADLE restored the hyperexcitability, an effect mediated by protein kinase C (PKC). DOPr-dependent DRG neuron hyperexcitability was blocked by the endocytosis inhibitor Pitstop 2 and the weakly internalizing DOPr agonist ARM390 did not cause hyperexcitability. BRET studies in HEK cells showed no evidence of switching of G-protein signaling from G to a G pathway in response to either high concentrations or overnight incubation of opioids. Thus, chronic high dose opioid exposure leads to OT and features of OIH in the colon. This action is mediated by DOPr signaling and is dependent on receptor endocytosis and downstream PKC signaling.Opioids are effective in the treatment of abdominal pain but escalating doses can lead to opioid tolerance (OT) and potentially opioid-induced hyperalgesia (OIH). We found that δ-opioid receptor (DOPr) plays a central role in the development of OT and OIH in colonic afferent nociceptors following prolonged exposure to high concentrations of MOPr or DOPr agonists. Furthermore, the role of DOPr was dependent on OPr internalization and activation of a protein kinase C (PKC) signaling pathway. Thus, targeting DOPr or key components of the downstream signaling pathway could mitigate adverse side effects by opioids.