The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca(2+)](i), Ca(2+) transients and membrane Ca(2+) current, I(Ca), in cultured murine HL-1 cardiomyocytes. LY294002 (1-20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca(2+)](i), Ca(2+) transients and I(Ca). We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2-8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca(2+)](i) regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca(2+)](i), and inhibited Ca(2+) transients. Triciribine (1-20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca(2+)](i), and Ca(2+) transients and I(Ca). We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca(2+)](i) in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca(2+)](i) required for excitation-contraction coupling in cardiomyoctyes.