Background— Mice lacking guanylyl cyclase-A (GC-A), a natriuretic peptide receptor, have pressure-independent cardiac hypertrophy. However, the mechanism underlying GC-A–mediated inhibition of cardiac hypertrophy remains to be elucidated. In the present report, we examined the role of regulator of G-protein signaling subtype 4 (RGS4), a GTPase activating protein for G q and G i , in the antihypertrophic effects of GC-A. Methods and Results— In cultured cardiac myocytes, treatment of atrial natriuretic peptide stimulated the binding of guanosine 3′,5′-cyclic monophosphate-dependent protein kinase (PKG) I-α to RGS4, PKG-dependent phosphorylation of RGS4, and association of RGS4 and Gα q . In contrast, blockade of GC-A by an antagonist, HS-142-1, attenuated the phosphorylation of RGS4 and association of RGS4 and Gα q . Moreover, overexpressing a dominant negative form of RGS4 diminished the inhibitory effects of atrial natriuretic peptide on endothelin-1–stimulated inositol 1,4,5-triphosphate production, [ 3 H]leucine incorporation, and atrial natriuretic peptide gene expression. Furthermore, expression and phosphorylation of RGS4 were significantly reduced in the hearts of GC-A knockout (GC-A-KO) mice compared with wild-type mice. For further investigation, we constructed cardiomyocyte-specific RGS4 transgenic mice and crossbred them with GC-A-KO mice. The cardiac RGS4 overexpression in GC-A-KO mice significantly reduced the ratio of heart to body weight ( P <0.001), cardiomyocyte size ( P <0.01), and ventricular calcineurin activity ( P <0.05) to 80%, 76%, and 67% of nontransgenic GC-A-KO mice, respectively. It also significantly suppressed the augmented cardiac expression of hypertrophy-related genes in GC-A-KO mice. Conclusions— These results provide evidence that GC-A activates cardiac RGS4, which attenuates Gα q and its downstream hypertrophic signaling, and that RGS4 plays important roles in GC-A–mediated inhibition of cardiac hypertrophy.