Abstract Body: Introduction: Cardiomyocyte maturation is a postnatal heart development that requires precise molecular, metabolic, structural, and electrophysiological changes to sustain the increased workload of the adult heart. Previous studies demonstrate that alternative RNA splicing is a key regulatory mechanism during this process. However, the full landscape of isoform switch and its role in functional specialization in adult cardiomyocyte remain largely unexplored. This study aims to provide a comprehensive landscape of isoform utilization during heart maturation. Methods: Using PacBio Iso-Seq, we profiled full-length transcriptomic changes in mouse left ventricles across neonatal to postnatal developmental stages. Isoform expression and usage ratios were analyzed to identify the genes with significant isoform switch. Functional validation of selected isoforms was performed in hESC-CMs. Results: While the total number of genes expressed remains relatively stable, the total number of full-length transcripts decreased from E18 to P49, indicating a progressive reduction in transcriptomic complexity at the isoform level. PCA analysis of isoform-specific expression and ratios shows stage-dependent clustering, highlighting that cardiac isoform dynamics is a defining feature for postnatal heart maturation. We identified at least 50 genes showing significant isoform switches across the maturation stages. In addition, an isoform of PDLIM5 is found to have exon 5B inclusion (PDLIM5-Ex5B) and its expression is increased in adult mouse heart but reversed to neonatal level in the mouse failing heart (n=3, p<0.05 compared to P49 and Sham control). hESC-CMs expressing PDLIM5-Ex5B improved sarcomere organization, enhanced binucleation, and increased expression of maturation markers. In contrast, isoforms lacking exon 5B were less effective in promoting cardiomyocyte maturation features. These findings further confirm that alternative splicing-mediated isoform switch is critical for cardiomyocyte structural and functional maturation. Conclusions: This study provides a comprehensive landscape of RNA splicing and isoform switching events during heart maturation, highlighting their importance in defining postnatal heart maturation stages and achieving functional specialization. Our results serve as a valuable resource for exploring maturation-related RNA splicing regulators and may represent a potential splicing-targeted therapy for heart diseases.