The calcium-responsive molecule, calcineurin, has been well characterized to play a causal role in pathological cardiac hypertrophy over the past decade. However, the intrinsic negative regulation of calcineurin signaling during the progression of cardiomyocyte hypertrophy remains enigmatic. Herein, we explored the role of EPI64C, a dual inhibitor of both Ras and calcineurin signaling during T-cell activation, in pressure overload-induced cardiac hypertrophy. We generated a cardiac-specific Epi64c conditional knockout mouse strain and showed that loss of Epi64c remarkably exacerbates pressure overload-induced cardiac hypertrophy. In contrast, EPI64C gain-of-function in cardiomyocytespecific Epi64c transgenic mice exerts potent protective effects against cardiac hypertrophy. Mechanistically, the cardioprotective effects of EPI64C are largely attributed to the disrupted calcineurin signaling but are independent of its Ras suppressive capability. Molecular studies have indicated that the 406 to 446 C-terminal amino acids in EPI64C directly bind to the 287 to 337 amino acids in the catalytic domain of calcineurin, which is responsible for the EPI64Cmediated suppressive effects. We further extrapolated our studies to cynomolgus monkeys and showed that gene therapy based on lentivirus-mediated EPI64C overexpression in the monkey hearts blunted pressure overload-induced cardiac hypertrophy. Our study thus identified EPI64C as a novel negative regulator in cardiac hypertrophy by targeting calcineurin signaling and demonstrated the potential of gene therapy and drug development for treating cardiac hypertrophy.