The phosphoinositide 3-kinase (PI3-kinase)-protein kinase B (Akt) signaling pathway is essential in the induction of physiological cardiac hypertrophy. In contrast, protein kinase C β2 (PKCβ2) is implicated in the development of pathological cardiac hypertrophy and heart failure. Thus far, no clear association has been demonstrated between these two pathways. In this study, we examined the potential interaction between the PI3-kinase and PKCβ2 pathways by crossing transgenic mice with cardiac specific expression of PKCβ2, constitutively active (ca) PI3-kinase, and dominant-negative (dn) PI3-kinase. In caPI3-kinase/PKCβ2 and dnPI3-kinase/PKCβ2 double-transgenic mice, the heart weight-to-body weight ratios and cardiomyocyte sizes were similar to those observed in caPI3-kinase and dnPI3-kinase transgenic mice, respectively, suggesting that the regulation of physiological developmental hypertrophy via modulation of cardiomyocyte size proceeds through the PI3-kinase pathway. In addition, we observed that caPI3-kinase/PKCβ2 mice showed improved cardiac function while the function of dnPI3-kinase/PKCβ2 mice was similar to that of the PKCβ2 group. PKCβ2 protein levels in both dnPI3-kinase/PKCβ2 and PKCβ2 mice were significantly upregulated. Interestingly, however, PKCβ2 protein expression was significantly attenuated in caPI3-kinase/PKCβ2 mice. PI3-kinase activity measured by Akt phosphorylation was not affected by PKCβ2 overexpression. These data suggest a potential interaction between these two pathways in the heart, where PI3-kinase is predominantly responsible for the regulation of physiological developmental hypertrophy and may act as an upstream modulator of PKCβ2 with the potential for rescuing the pathological cardiac dysfunction induced by overexpression of PKCβ.