Reversible airway constriction is induced by an increase in airway smooth muscle contractility in response to methacholine likely as a bronchospastic stimulus. Despite the finding of Galpha12 and Galpha13 up-regulation in airway hyperresponsive animals, their functional role of contraction in airway smooth muscle has not been directly explored. This study investigated the differential regulatory role of Galpha12/Galpha13 in methacholine-induced contraction of trachea and bronchus in Galpha12 or Galpha13 gene knockout mice after ovalbumin sensitization and challenges. Organ bath assays and videomicroscopy revealed that Galpha13 deficiency delayed methacholine-induced contractile response of bronchiolar smooth muscle, but not that of tracheal smooth muscle. In primary bronchial smooth muscle cells, knockdown of Galpha13 blocked methacholine-induced phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20), a prerequisite step for the contractile initiation of actin and myosin. Galpha13-dependent MLC20 phosphorylation was confirmed in murine embryonic fibroblasts. After ovalbumin sensitization and challenges, wild type mice exhibited methacholine-induced bronchial contraction of lung tissue. Heterozygous absence of the Galpha13 gene abrogated methacholine-induced contractions, whereas homozygous absence of the Galpha12 gene failed to do so. Our findings indicate that Galpha13, but not Galpha12, specifically regulates cholinergic bronchial contraction in airway responsiveness via controlling phosphorylation of MLC20 by methacholine.