Two major β-adrenergic receptor (βAR) subtypes, β(1)AR and β(2)AR, are expressed in mammalian heart with β(1)AR coupling to G(s) and β(2)AR dually coupling to G(s) and G(i) proteins. In many types of chronic heart failure, myocardial contractile response to both β(1)AR and β(2)AR stimulation is severely impaired. The dysfunction of βAR signaling in failing hearts is largely attributable to an increase in G(i) signaling, because disruption of the G(i) signaling restores myocardial contractile response to β(1)AR as well as β(2)AR stimulation. However, the mechanism terminating the β(2)AR-G(i) signaling remains elusive, while it has been shown activation of the G(i) signaling is dependent on agonist stimulation and subsequent PKA-mediated phosphorylation of the receptor. Here we demonstrate that regulator of G protein signaling 2 (RGS2) is a primary terminator of the β(2)AR-G(i) signaling. Specifically, prolonged absence of agonist stimulation for 24h impairs the β(2)AR-G(i) signaling, resulting in enhanced β(2)AR- but not β(1)AR-mediated contractile response in cultured adult mouse cardiomyocytes. Increased β(2)AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3-5) or G(s), G(i) or βAR subtypes. Administration of a βAR agonist, isoproterenol (ISO, 1.0 nM), prevents RGS2 upregulation and restores the β(2)AR-G(i) signaling in cultured cells. Furthermore, RGS2 ablation, similar to βAR agonist stimulation, sustains the β(2)AR-G(i) signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated β(2)AR-G(i) signaling in cardiomyocytes and HEK293 cells. These findings not only define RGS2 as a novel negative regulator of the β(2)AR-G(i) signaling but also provide a potential novel target for the treatment of chronic heart failure.