Gastrointestinal (GI) dysfunction is commonly reported by people diagnosed with autism spectrum disorder (ASD; autism) but the cause is unknown. Mutations in genes encoding synaptic proteins including Neuroligin-3 are associated with autism. Mice lacking Neuroligin-3 (Nlgn3 ) have altered brain function, but whether the enteric nervous system (ENS) is altered remains unknown. We assessed for changes in GI structure and function in Nlgn3 mice. We found no significant morphological differences in villus height or crypt depth in the jejunum or colon between wildtype (WT) and Nlgn3 mice. To determine whether deletion of Nlgn3 affects enteric neurons, we stained for neural markers in the myenteric plexus. Nlgn3 mice had similar numbers of neurons expressing the pan-neuronal marker Hu in the jejunum, proximal mid, and distal colon regions. We also found no differences in the number of neuronal nitric oxide synthase (nNOS+) or calretinin (CalR+) motor neurons and interneurons between WT and Nlgn3 mice. We used ex vivo video imaging analysis to assess colonic motility under baseline conditions and observed faster colonic migrating motor complexes (CMMCs) and an increased colonic diameter in Nlgn3 mice, although CMMC frequency was unchanged. At baseline, CMMCs were faster in Nlgn3 mice compared to WT. Although the numbers of neuronal subsets are conserved in Nlgn3 mice, these findings suggest that Neuroligin-3 modulates inhibitory neural pathways in the ENS and may contribute to mechanisms underlying GI disorders in autism. Autism Res 2020, 13: 691-701. © 2019 The Authors. Autism Research published by International Society for Autism Research published byWiley Periodicals, Inc. LAY SUMMARY: People with autism commonly experience gut problems. Many gene mutations associated with autism affect neuronal activity. We studied mice in which the autism-associated Neuroligin-3 gene is deleted to determine whether this impacts gut neuronal numbers or motility. We found that although mutant mice had similar gut structure and numbers of neurons in all gut regions examined, they had distended colons and faster colonic muscle contractions. Further work is needed to understand how Neuroligin-3 affects neuron connectivity in the gastrointestinal tract.