Methacholine (MCH) is an agonist that can bind to airway smooth muscle to induce bronchoconstriction and consequently labored breathing. Previous research has shown that MCH induces labored breathing in a mouse model of adolescent asthma. Here, we show that methacholine is associated with an acute inflammatory response in mice, measured by heightened inflammatory cytokine gene expression. This association between MCH and acute inflammation will have applications for future asthma studies utilizing methacholine.