Interferon regulatory factor (IRF) 3, a member of the highly conserved IRF family transcription factors, plays a pivotal role in innate immune response, apoptosis, and oncogenesis. Recent studies have implicated IRF3 in a wide range of host defense. However, whether IRF3 induces defensive responses to hypertrophic stresses such as biomechanical stress and neurohumoral factors remains unclear. Herein, we employed an IRF3-deficient mouse model, cardiac-specific IRF3-overexpression mouse model and isolated cardiomyocytes to investigate the role of IRF3 in cardiac hypertrophy induced by aortic banding (AB) or isoproterenol (ISO). The extent of cardiac hypertrophy was quantitated by echocardiography as well as by pathological and molecular analysis. Our results demonstrate that IRF3 deficiency profoundly exacerbated cardiac hypertrophy, whereas overexpression of IRF3 in the heart significantly blunted pathological cardiac remodeling induced by pressure overload. Similar results were also observed in cultured cardiomyocytes upon the treatment with ISO. Mechanistically, we discovered that IRF3 interacted with ERK2 and thereby inhibited the ERK1/2 signaling. Furthermore, inactivation of ERK1/2 by U0126 offset the IRF3-deficient-mediated hypertrophic response induced by aortic banding. Altogether, these data demonstrate that IRF3 plays a protective role in AB-induced hypertrophic response by inactivating ERK1/2 in the heart. Therefore, IRF3 could be a new target for the prevention and therapy of cardiac hypertrophy and failure.