Cardiac hypertrophy is a compensatory response to pressure overload, leading to heart failure. Recent studies have demonstrated crucial roles of Rho and its downstream proteins in cardiac hypertrophic responses. However, the detail mechanisms how Rho signaling controls hypertrophic responses remain incompletely understood. Here, we show that mammalian homolog of Drosophila diaphanous (mDia) 1, a Rho-effector molecule, plays pivotal roles in pressure overload-induced ventricular hypertrophy. mDia1-knockout (mDia1KO) mice subjected to transverse aortic constriction (TAC) exhibited attenuated cardiac hypertrophic responses in the early phase and poor survival in the chronic phase. Microarray analysis revealed that mDia1 was involved in the induction of hypertrophy related genes targeted by the serum response factor (SRF) pathway, e.g. immediate early genes, in pressure overloaded hearts. Loss of mDia1 attenuated the activation of focal adhesion kinase and extracellular signal-regulated kinase in both TAC-operated mice hearts and mechanical stretched neonatal rat ventricular cardiomyocytes. Furthermore, the increase in the F/G-actin ratio induced by TAC was suppressed in mDia1KO hearts, thereby resulted in a decrease in the nuclear accumulation of myocardin-related transcription factors and SRF. Our data demonstrate that mDia1 coordinates signal transduction triggered by pressure overload through the regulation of actin homeostasis, which is required for proper cardiac responses to pathological stimuli.