Tiotropium, a long-acting anticholinergic, may improve chronic obstructive pulmonary disease (COPD) by mechanisms beyond bronchodilatation. We tested the hypothesis that tiotropium may act as an anti-inflammatory mediator by directly acting on and inhibiting human neutrophil chemotactic activity (NCA) that is promoted by acetylcholine (ACh) exposure. ACh treatment increased NCA in a dose dependent manner (p < 0.001) and tiotropium pretreatment reduced ACh stimulation (dose effect; 0 to 1000 nM; p < 0.001). Selective muscarinic receptor inhibitors demonstrated that subtype-3 (M3) receptor plays a role in NCA regulation. In addition, NCA that was stimulated by cevimeline (M3 agonist) and pasteurella multocida toxin (PMT, M3 coupled Gq agonist). However, the increased NCA to cevimeline and PMT was reduced by tiotropium pretreatment (p < 0.001). ACh treatment stimulated ERK-1/2 activation by promoting protein phosphorylation and tiotropium reduced this effect (p < 0.01). In addition, pretreatment of the cells with a specific MEK-1/2 kinase inhibitor reduced ACh stimulated NCA (p < 0.01). Together these results demonstrated that cholinergic stimulation of NCA is effectively inhibited by tiotropium and is governed by a mechanism involving M3 coupled Gq signaling and downstream ERK signaling. This study further demonstrates that tiotropium may act as an anti-inflammatory agent in lung disease.