The enteric nervous system (ENS) controls digestion autonomously via a complex neural network within the gut wall. Enteric neurons expressing glutamate have been identified by transcriptomic studies as a distinct subpopulation, and glutamate can affect intestinal motility by modulating enteric neuron activity. However, the nature of glutamatergic neurons, their position within the ENS circuit, and their function in regulating gut motility are unknown. We identify glutamatergic neurons as longitudinally projecting descending interneurons in the small intestine and colon and as a novel class of circumferential neurons only in the colon. Both populations make synaptic contact with diverse neuronal subtypes and signal with multiple neurotransmitters and neuropeptides in addition to glutamate, including acetylcholine and enkephalin. Knocking out the glutamate transporter VGLUT2 from enkephalin neurons disrupts gastrointestinal transit, while ex vivo optogenetic stimulation of glutamatergic neurons initiates colonic propulsive motility. Our results posit glutamatergic neurons as key interneurons that regulate intestinal motility.