The deacetylase Sirtuin-1 (Sirt1) is involved in the cardiac hypertrophic responses and cardiac embryo morphogenesis. However, the physiological function of Sirt1 deficiency in the postnatal development of the heart remains to be characterized. The aim of the study was to investigate the relevance of Sirt1 in the development and function of the myocardium. Hearts from Sirt1-deficient mice partially or totally lacking Sirt1 protein activity were analyzed. Loss of Sirt1 activity led to dilated cardiomyopathy in adult hearts, a phenotype accompanied by reduced cardiomyocyte size and the absence of fibrosis. Morphological and functional mitochondrial abnormalities were observed in the adult hearts lacking Sirt1, suggesting that mitochondrial dysfunction contributes to the progression of the observed cardiomyopathy. Moreover, gene expression analyses revealed that mitochondrial genes were the most affected in Sirt1-deficient mice, showing a reduction in their expression. No overt cardiac dilatation was observed in neonates lacking Sirt1 activity, but first signs of mitochondrial alterations were already present. Immunoblot analyses revealed that Sirt1 is highly expressed in the heart after birth, indicating the importance of Sirt1 in the neonatal period. Finally, Sirt1 deficiency affected the acetylation pattern of the myocyte enhancer factor 2 (Mef2) transcription factors, which are critical for normal heart development and mitochondrial integrity. Collectively, our findings indicate that Sirt1 is essential for the maintenance of cardiac mitochondrial integrity and normal postnatal myocardium development.