Epigenetic modifications have emerged as central players in the coordination of gene expression networks during cardiac development. Much attention has focused on the role of histone modifications during embryonic heart development, but relatively little is known about the epigenetic control mechanisms that guide post-natal heart maturation. Furthermore, few studies have investigated the role of DNA methylation during cardiac development, despite the fundamental importance of this biological process for transcriptional regulation. The purpose of the current study was to determine whether DNA methylation plays an important role in guiding transcription during the neonatal period, which is an important developmental window for cardiac maturation, including cardiomyocyte cell cycle withdrawal and loss of endogenous regenerative capacity. Here, we interrogated genome-wide changes in transcription and CpG methylation during post-natal cardiac maturation in the mouse (P1 vs. P14). CpG sequencing (CpG-seq) identified 2251 differentially methylated regions (DMRs) between P1 and P14. Intersection of DMRs with different genomic features revealed that 1248 DMRs were associated with promoter regions and transcription start sites. Interestingly, increased methylation of genes associated with well-known signalling pathways for muscle development and differentiation, such as the bone morphogenetic protein, fibroblast growth factor, Wnt and Notch signalling pathways, was associated with transcriptional repression of these regulatory networks at P14. To determine the functional significance of these dynamic changes in the cardiac methylome, we inhibited DNA methylation in vivo by administration of the DNA hypomethylating agent 5-azacytidine from P2 to P12. Post-natal inhibition of DNA methylation caused a marked increase in heart size and was associated with increased cardiomyocyte proliferation. This study provides evidence for widespread alterations in DNA methylation during post-natal heart maturation and suggests that DNA methylation may play an important role in the transcriptional silencing of key regulatory networks for muscle development and cardiomyocyte proliferation during neonatal life.