Sunitinib (SNT) is a multi-targeted receptor tyrosine kinase inhibitor that has been approved by the FDA for cancer therapy. However, its cardiotoxicity has limited the clinical applicability with no effective therapeutic approach available. As a broadband kinase inhibitor, the function of several kinases that are essential to cardiac function might also be affected by SNT, such as calmodulin-dependent protein kinase (CaMKII), cyclic-AMP-dependent protein kinases (PKA), AMP-activated protein kinase (AMPK), and phosphoinositide 3 kinase (PI3K). In this study, we investigated whether SNT-induced cardiotoxicity could be prevented by blocking SNT-induced alteration in the corresponding signaling pathways. In human induced pluripotent stem cell-derived cardiomyocytes, SNT (0.5-20 µmol/L) inhibited contractility of cardiomyocytes in a concentration-dependent manner, and the inhibitory effect was prevented either by PIP3 (1 µmol/L) application or PI3K overexpression. On the contrary, the CaMKII inhibitor KN-93 (50 nmol/L), PKA inhibitor H89 (1 µmol/L), and AMPK activators metformin (2 mmol/L) and 5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside (2 mmol/L) presented negligible effects. Oral SNT administration (40 mg/kg/day) in mice progressively decreased the PI3K activity and cardiac function in 2 weeks with a significant decrease in the expression and activity of Cav1.2 and SERCA. Cardiac-specific PI3K overexpression through adeno-associated virus 9-mediated gene delivery in mice prevented SNT-induced reduction in cardiac function, calcium transient, calcium current, and Cav1.2 expression. In summary, our data indicate that increased PI3K activity is protective against SNT-induced calcium mishandling and contractile dysfunction. Cardiac-specific PI3K activation could be an effective therapeutic approach to treat SNT cardiotoxicity in patients with cancer.