The μ-opioid receptor (MOR) is a major regulator of gastrointestinal motility and secretion and mediates opiate-induced bowel dysfunction. Although MOR is of physiological and therapeutic importance to gut function, the cellular and subcellular distribution and regulation of MOR within the enteric nervous system are largely undefined. Herein, we defined the neurochemical coding of MOR-expressing neurons in the guinea pig gut and examined the effects of opioids on MOR trafficking and regulation. MOR expression was restricted to subsets of enteric neurons. In the stomach MOR was mainly localized to nitrergic neurons (∼88%), with some overlap with neuropeptide Y (NPY) and no expression by cholinergic neurons. These neurons are likely to have inhibitory motor and secretomotor functions. MOR was restricted to noncholinergic secretomotor neurons (VIP-positive) of the ileum and distal colon submucosal plexus. MOR was mainly detected in nitrergic neurons of the colon (nitric oxide synthase positive, 87%), with some overlap with choline acetyltransferase (ChAT). No expression of MOR by intrinsic sensory neurons was detected. [d-Ala², MePhe⁴, Gly(ol)⁵]enkephalin (DAMGO), morphiceptin, and loperamide induced MOR endocytosis in myenteric neurons. After stimulation with DAMGO and morphiceptin, MOR recycled, whereas MOR was retained within endosomes following loperamide treatment. Herkinorin or the δ-opioid receptor agonist [d-Ala², d-Leu⁵]enkephalin (DADLE) did not evoke MOR endocytosis. In summary, we have identified the neurochemical coding of MOR-positive enteric neurons and have demonstrated differential trafficking of MOR in these neurons in response to established and putative MOR agonists.