Background— Diacylglycerol is a lipid second messenger that accumulates in cardiomyocytes when stimulated by Gqα protein-coupled receptor (GPCR) agonists such as angiotensin II, phenylephrine, and others. Diacylglycerol functions as a potent activator of protein kinase C (PKC) and is catalyzed by diacylglycerol kinase (DGK) to form phosphatidic acid and inactivated. However, the functional roles of DGK have not been previously examined in the heart. We hypothesized that DGK might prevent GPCR agonist-induced activation of diacylglycerol downstream signaling cascades and subsequent cardiac hypertrophy. Methods and Results— To test this hypothesis, we generated transgenic (DGKζ-TG) mice with cardiac-specific overexpression of DGKζ. There were no differences in heart size and heart weight between DGKζ-TG and wild-type littermate mice. The left ventricular function was normal in DGKζ-TG mice. Continuous administration of subpressor doses of angiotensin II and phenylephrine caused PKC translocation, gene induction of atrial natriuretic factor, and subsequent cardiac hypertrophy in WT mice. However, in DGKζ-TG mice, neither translocation of PKC nor upregulation of atrial natriuretic factor gene expression was observed after angiotensin II and phenylephrine infusion. Furthermore, in DGKζ-TG mice, angiotensin II and phenylephrine failed to increase cross-sectional cardiomyocyte areas and heart to body weight ratios. Phenylephrine-induced increases in myocardial diacylglycerol levels were completely blocked in DGKζ-TG mouse hearts, suggesting that DGKζ regulated PKC activity by controlling cellular diacylglycerol levels. Conclusions— These results demonstrated the first evidence that DGKζ negatively regulated the hypertrophic signaling cascade and resultant cardiac hypertrophy in response to GPCR agonists without detectable adverse effects in in vivo hearts.