Colon epithelial cells express and most colon cancers overexpress M(3) muscarinic receptors (M(3)R). In human colon cancer cells, post-M(3)R signaling stimulates proliferation. To explore the importance of M(3)R expression in vivo, we used the azoxymethane-induced colon neoplasia model. Mice treated with weekly i.p. injection of saline [10 wild-type (WT) mice] or azoxymethane (22 WT and 16 M(3)R(-/-) mice) for 6 weeks were euthanized at 20 weeks. At week 20, azoxymethane-treated WT mice weighed approximately 16% more than M(3)R(-/-) mice (33.4 grams +/- 1.0 grams versus 27.9 grams +/- 0.5 grams; mean +/- SE, P < 0.001). In azoxymethane-treated M(3)R(-/-) mice, cell proliferation (BrdUrd staining) was reduced 43% compared with azoxymethane-treated WT mice (P < 0.05). Whereas control mice (both WT and M(3)R(-/-)) had no colon tumors, azoxymethane-treated WT mice had 5.3 +/- 0.5 tumors per animal. Strikingly, azoxymethane-treated M(3)R(-/-) mice had only 3.2 +/- 0.3 tumors per mouse (P < 0.05), a 40% reduction. Tumor volume in azoxymethane-treated M(3)R(-/-) mice was reduced 60% compared with azoxymethane-treated WT mice (8.1 mm(3) +/- 1.5 mm(3) versus 20.3 mm(3) +/- 4.1 mm(3); P < 0.05). Compared with WT, fewer M(3)R(-/-) mice had adenomas (6% versus 36%; P = 0.05), and M(3)R(-/-) mice had fewer adenocarcinomas per mouse (0.6 +/- 0.1 versus 1.7 +/- 0.4; P < 0.05). Eleven of 22 WT but no M(3)R(-/-) mice had multiple adenocarcinomas (P < 0.001). Compared with WT, azoxymethane-treated M(3)R-deficient mice have attenuated epithelial cell proliferation, tumor number, and size. M(3)R and post-M(3)R signaling are novel therapeutic targets for colon cancer.