Glycogen synthase kinase-3 (GSK-3) is a known negative regulator of cardiac hypertrophic response and much remains unknown about its dephosphorylation process that activates GSK-3. Since GSK-3 function is regulated by PI3K, age based assessment of cardiac hypertrophic response was measured in PI3Kγ knockout (PI3Kγ KO) mice. Interestingly, we observed marked reduction in heart size in PI3Kγ KO mice compared to littermate controls. Consistent with the reduced size, we observed elevated PP2A activity and dephosphorylation mediated activation of GSK-3 in PI3Kγ-KO hearts. Mechanistically, we found that higher PP2A activity in PI3Kγ-KO was due to PP2A methylation mediated by elevated PP2A methyl transferase (PPMT-1) activity. To test in vivo whether PI3K activity regulates GSK-3 dephosphorylation mediated cardiac growth, we generated mice with cardiac specific overexpression of inactive PI3Kγ (PI3Kγinact) and constitutively active PI3Kγ (myrPI3Kγ) in the PI3Kγ-KO background. Age based hypertrophic response and cardiac function was assessed by heart weight/body weight ratios and echocardiography. The heart size was significantly increased in PI3Kγinact (LVEDD- 3.41+/- 0.12) and myrPI3Kγ (LVEDD- 3.73 +/- 0.14) overexpressing mice, when compared to WT (LVEDD- 3.00 +/- 0.15) mice at 18 months age, indicating that kinase activity of PI3Kγ is inconsequential for age-dependent regulation of cardiac growth. Similar cardiac growth due to overexpression of active and inactive form of PI3Kγ suggests that the kinase independent function potentially overrides the kinase activity of PI3Kγ. Correspondingly, PP2A activity was normalized and was associated with decreased GSK-3 dephosphorylation with overexpression of PI3Kγinact. This led to normalization of heart size in these mice compared to PI3Kγ-KO littermates correlating with the decreased GSK-3 dephosphorylation and consequent inhibition of GSK-3 activity. Regulation of PP2A mediated dephosphorylation of GSK-3 by PI3Kγ combined with similar level of increased cardiac growth in PI3Kγ-KO mice with cardiac overexpression of PI3Kγinact or myrPI3Kγ, suggests a non-canonical role of PI3Kγ in hypertrophy of the heart due to age related increase in mechanical demand.