Expressed in a wide array of mammalian organs and tissues, acetylcholine receptors are categorized structurally and functionally as either muscarinic or nicotinic. Muscarinic receptors are further sub-categorized into five subtypes, designated M1R–M5R. As members of the guanine nucleotide binding (G) protein-coupled receptor superfamily muscarinic receptors transmit signals by activating G proteins that regulate downstream protein kinase cascades that alter gene expression or otherwise modulate cell function. Coupling to G proteins further distinguishes receptor subtypes as either G_q/11-bound (M1R, M3R, M5R) or G_i/o-bound (M2R, M4R). Gastrointestinal epithelial cells express M1R and M3R primarily. Herein, we focus on exploring functional differences between M1R and M3R activation, particularly with respect to responses in normal epithelium and different gastrointestinal cancers. Variable responses may result from allosteric binding sites, bitopic ligands, differential receptor distribution, and sequence variations that modulate the affinity and stability of ligand binding. Notably, major dissimilarities in the progression of gastrointestinal pathology result from M1R versus M3R expression or activation. Autocrine and paracrine M3R activation or M3R overexpression promotes neoplasia in the stomach and colon; blocking M3R expression or activation attenuates cancer progression. In contrast, M1R activation appears to act as a tumor suppressor; knockout of both M1R and M3R expression negate the anti-colon tumor effects of M3R knockout alone. Dynamic interplay between M1R and M3R activation and distinct post-receptor signaling in the gastrointestinal tract offers novel therapeutic possibilities. A more complete understanding of muscarinic receptor subtype function and action will likely result in innovative approaches to treating gastrointestinal disorders, including cancer.