The mu 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 is 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 NPY and no expression by cholinergic neurons. These neurons are likely to have inhibitory motor and secretomotor functions. MOR was restricted to non-cholinergic secretomotor neurons (VIP-positive) of the ileum and distal colon submucosal plexus. MOR was mainly detected in nitrergic neurons of the colon (NOS-positive, 87%), with some overlap with ChAT. No expression of MOR by intrinsic sensory neurons was detected. 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 delta opioid receptor agonist 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.