β₂-Adrenergic receptors (β₂AR) are expressed on airway smooth muscle cells and act to relax the airway on activation by β-agonists. These agents are utilized for treating asthma but are associated with adverse outcomes. To ascertain the effects of persistent β₂AR activation on gene expression, cultured airway smooth muscle cells derived from wild-type (WT) and transgenic mice overexpressing β₂AR were subjected to DNA microarray analysis; 319 genes were increased and 164 were decreased. Differential expression was observed in genes from 22 Gene Ontology Slim categories, including those associated with ion transport and calcium ion binding. A 60% decrease (P = 0.008) in phospholamban (PLN), an intracellular Ca²⁺ concentration ([Ca²⁺]_i)-handling protein that is at a signaling nodal point in cardiomyocytes, was observed in β₂AR-overexpressing cells and confirmed at the protein level. To isolate the physiological effect of decreased PLN in airway smooth muscle, airway contraction and relaxation responses were studied in WT and PLN^−/− mice. PLN^−/− mice had a markedly reduced constrictive response to methacholine. In contrast, the bronchodilatory effect of β-agonist was not different between WT and PLN^−/− mice. These results revealed an unanticipated therapeutic effect of β-agonists, PLN downregulation, which acts to decrease airway hyperreactivity. Thus agents that inhibit PLN may act synergistically with the bronchodilating action of β-agonists. A number of other genes related to [Ca²⁺]_i are also differentially regulated by β₂AR activity, some of which may act to oppose, or augment, the efficacy of chronic β-agonists. These genes or pathways may also represent additional targets in the treatment of asthma and related obstructive lung diseases.