BACKGROUND Intestinal mucosal homeostasis is controlled by multiple factors and an intact, functional mucosa is essential for survival. Maintenance of the epithelium begins with crypt base stem cells which eventually give rise to all epithelial cell types. Evidence suggests an important role of the enteric cholinergic nervous system in these processes. We hypothesized that mice with altered muscarinic signaling would exhibit differences in mucosal morphometric and proliferative parameters compared to wild-type mice. METHODS Mouse lines specifically deficient in one of the five muscarinic acetylcholine receptors (M1KO-M5KO) were used for experiments. Distal ileal segments were obtained and histologic sections created. Villus height and crypt depth were measured using H&E-stained sections, while crypt proliferation index (CPI) was calculated using Ki67-stained sections. RESULTS The ileal mucosa from mice deficient in mAChRs exhibited differences from wild-type ileal mucosa in nearly all measured parameters. Knockout of mAChR2, mAChR3 and mAChR5 resulted in changes in all measured parameters. Ileal mucosa from M2KO mice showed an unexpected combination decreased VH but paradoxically increased CD and CPI. CONCLUSIONS Alterations in mAChR signaling causes change in ileal mucosal morphometry and crypt cell proliferation. While all mAChR subtypes may be involved, mAChR2, mAChR3, and mAChR5 appear to be critical for mucosal homeostasis. Further characterization of these pathways is warranted.