Abstract Body: Although phosphoinositide 3-kinase (PI3Kγ) null mice (PI3Kγ^-/-) show increased ventricular rate/contraction, it is unknown whether PI3Kγ regulates calcium recycling machinery underlying this phenotype. Primary adult cardiomyocytes from PI3Kγ^-/- mice show altered sarcoendoplasmic reticulum (SR) calcium cycling following caffeine. Unexpectedly, PI3Kγ^-/- cardiomyocytes showed significant reduction in phosphorylation of phospholamban (PLN) at Thr17, a key regulator of SR calcium re-uptake without changes in phosphorylation at Ser16. Furthermore, loss in PLN phosphorylation in PI3Kγ^-/- cardiomyocytes was associated with augmented interaction with SR calcium ATPase (SERCA). Surprisingly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ_inact) in global PI3Kγ^-/- mice (PI3Kγ_inact/PI3Kγ^-/-) normalized caffeine-induced calcium re-uptake, PLN phosphorylation at Thr17 and decreased PLN-SERCA interaction. These data suggested kinase-independent function of PI3Kγ in regulation of SR calcium load and PLN phosphorylation. Since phosphorylation of Thr17 of PLN is carried out by Ca²⁺/Calmodulin dependent protein kinase (CamKII), we probed for the role of PI3Kγ in regulation of CamKII in PLN phosphorylation. Mechanistically, PI3Kγ exhibits scaffolding function in recruitment of CamKII to PLN at SR to mediate PLN phosphorylation. Furthermore, we showed that PI3Kγ directly interacts with CamKII. The study unravels a yet to be recognized kinase-independent role of PI3Kγ in regulating PLN with implications in cardiac function.