Colon epithelial cells express and most colon cancers overexpress M₃ muscarinic receptors (M₃R). In human colon cancer cells, post-M₃R signaling stimulates proliferation. To explore the importance of M₃R 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 M₃R^−/− mice) for 6 weeks were euthanized at 20 weeks. At week 20, azoxymethane-treated WT mice weighed ∼16% more than M₃R^−/− mice (33.4 grams ± 1.0 grams versus 27.9 grams ± 0.5 grams; mean ± SE, P < 0.001). In azoxymethane-treated M₃R^−/− mice, cell proliferation (BrdUrd staining) was reduced 43% compared with azoxymethane-treated WT mice (P < 0.05). Whereas control mice (both WT and M₃R^−/−) had no colon tumors, azoxymethane-treated WT mice had 5.3 ± 0.5 tumors per animal. Strikingly, azoxymethane-treated M₃R^−/− mice had only 3.2 ± 0.3 tumors per mouse (P < 0.05), a 40% reduction. Tumor volume in azoxymethane-treated M₃R^−/− mice was reduced 60% compared with azoxymethane-treated WT mice (8.1 mm³ ± 1.5 mm³ versus 20.3 mm³ ± 4.1 mm³; P < 0.05). Compared with WT, fewer M₃R^−/− mice had adenomas (6% versus 36%; P = 0.05), and M₃R^−/− mice had fewer adenocarcinomas per mouse (0.6 ± 0.1 versus 1.7 ± 0.4; P < 0.05). Eleven of 22 WT but no M₃R^−/− mice had multiple adenocarcinomas (P < 0.001). Compared with WT, azoxymethane-treated M₃R-deficient mice have attenuated epithelial cell proliferation, tumor number, and size. M₃R and post-M₃R signaling are novel therapeutic targets for colon cancer. [Cancer Res 2008;68(10):3573–8]