β₂AR desensitization in airway smooth muscle (ASM) mediated by airway inflammation has been proposed to contribute to asthma pathogenesis and diminished efficacy of β-agonist therapy. Mechanistic insight into this phenomenon is largely conceptual and lacks direct empirical evidence. Here, we employ molecular and genetic strategies to reveal mechanisms mediating cytokine effects on ASM β₂AR responsiveness. Ectopic expression of inhibitory peptide (PKI-GFP) or a mutant regulatory subunit of PKA (RevAB-GFP) effectively inhibited intracellular PKA activity in cultured human ASM cells and enhanced β₂AR responsiveness by mitigating both agonist-specific (β-agonist-mediated) desensitization and cytokine (IL-1β and TNF-α)-induced heterologous desensitization via actions on multiple targets. In the absence of cytokine treatment, PKA inhibition increased β₂AR-mediated signaling by increasing both β₂AR−G protein coupling and intrinsic adenylyl cyclase activity. PKI-GFP and RevAB-GFP expression also conferred resistance to cytokine-promoted β₂AR−G protein uncoupling and disrupted feed-forward mechanisms of PKA activation by attenuating the induction of COX-2 and PGE₂. Cytokine treatment of tracheal ring preparations from wild-type mice resulted in a profound loss of β-agonist-mediated relaxation of methacholine-contracted rings, whereas rings from EP2 receptor knockout mice were largely resistant to cytokine-mediated β₂AR desensitization. These findings identify EP2 receptor- and PKA-dependent mechanisms as the principal effectors of cytokine-mediated β₂AR desensitization in ASM.