Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of β₂-adrenoceptor (β₂-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of β₂-AR-coupled G_i proteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the β₂-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the β₂-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables β₂-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in β₂-AR-induced cAMP formation. Blocking G_i or Gβγ signaling with pertussis toxin or βARK-ct, a peptide inhibitor of Gβγ, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of β₂-AR-PKA signaling sequentially involves G_i, Gβγ, and PI3K. Thus, PI3K constitutes a key downstream event of β₂-AR-G_i signaling, which confines and negates the concurrent β₂-AR/G_s-mediated PKA signaling.