Anticholinergics, blocking the muscarinic M₃ receptor, are effective bronchodilators for patients with chronic obstructive pulmonary disease. Recent evidence from M₃ receptor-deficient mice (M₃R^−/−) indicates that M₃ receptors also regulate neutrophilic inflammation in response to cigarette smoke (CS). M₃ receptors are present on almost all cell types, and in this study we investigated the relative contribution of M₃ receptors on structural cells vs. inflammatory cells to CS-induced inflammation using bone marrow chimeric mice. Bone marrow chimeras (C56Bl/6 mice) were generated, and engraftment was confirmed after 10 wk. Thereafter, irradiated and nonirradiated control animals were exposed to CS or fresh air for four consecutive days. CS induced a significant increase in neutrophil numbers in nonirradiated and irradiated control animals (4- to 35-fold). Interestingly, wild-type animals receiving M₃R^−/− bone marrow showed a similar increase in neutrophil number (15-fold). In contrast, no increase in the number of neutrophils was observed in M₃R^−/− animals receiving wild-type bone marrow. The increase in keratinocyte-derived chemokine (KC) levels was similar in all smoke-exposed groups (2.5- to 5.0-fold). Microarray analysis revealed that fibrinogen-α and CD177, both involved in neutrophil migration, were downregulated in CS-exposed M₃R^−/− animals receiving wild-type bone marrow compared with CS-exposed wild-type animals, which was confirmed by RT-qPCR (1.6–2.5 fold). These findings indicate that the M₃ receptor on structural cells plays a proinflammatory role in CS-induced neutrophilic inflammation, whereas the M₃ receptor on inflammatory cells does not. This effect is probably not mediated via KC release, but may involve altered adhesion and transmigration of neutrophils via fibrinogen-α and CD177.