Cardiomyocyte elongation and alignment, a critical step in cardiomyocyte maturation starting from the perinatal stage, is crucial for formation of the highly organized intra- and inter-cellular structures for spatially and temporally ordered contraction in adult cardiomyocytes. However, the mechanism(s) underlying the control of cardiomyocyte alignment remains elusive. Here, we report that SIRT1, the most conserved NAD+-dependent protein deacetylase highly expressed in perinatal heart, plays an important role in regulating cardiomyocyte remodeling during development. We observed that SIRT1 deficiency impairs the alignment of cardiomyocytes/myofibrils and disrupts normal beating patterns at late developmental stages in an in vitro differentiation system from human embryonic stem cells. Consistently, deletion of SIRT1 at a late developmental stage in mouse embryos induced the irregular distribution of cardiomyocytes and misalignment of myofibrils, and reduced the heart size. Mechanistically, the expression of several genes involved in chemotaxis, including those in the CXCL12/CXCR4 and CCL2/CCR2/CCR4 pathways, was dramatically blunted during maturation of SIRT1-deficient cardiomyocytes. Pharmacological inhibition of CCL2 signaling suppressed cardiomyocyte alignment. Our study identifies a regulatory factor that modulates cardiomyocyte alignment at the inter-cellular level during maturation.