BACKGROUND: Mature mammalian cardiomyocytes (CMs) develop compact sarcomeric structures that inhibit proliferation. Consequently, CMs must dedifferentiate to a fetus-like state, which is accompanied by sarcomere disassembly, to enable successful cytokinesis. However, the regulation and coordination of CM dedifferentiation, cell cycle progression, and sarcomere reorganization remain unclear. METHODS: We generated adenovirus and adeno-associated virus (MyoAAV) vectors expressing YAP5SA and YAP5SA-S94A under X on control for LMI070-inducible protein expression. We also developed MyoAAV-cTnT- Tuba1b -shRNA- miR30 for cardiomyocyte-specific knockdown of Tuba1b . These tools were used to investigate CM dedifferentiation and proliferation and sarcomere disassembly. We also performed Cleavage Under Targets and Release Using Nuclease to map the genome-wide binding sites of YAP5SA and YAP5SA-S94A in combination with RNA sequencing to identify YAP target genes. In addition, time-course live-imaging analysis was used to evaluate microtubule and sarcomere dynamics in adult CMs. RESULTS: We show that microtubule expression and network density decline with cardiac maturation. Overexpression of YAP5SA, a constitutively active YAP mutant, promotes microtubule growth by stabilizing microtubule dynamics, leading to CM dedifferentiation, cell cycle re-entry, and sarcomere disassembly. In contrast, colchicine blocks these processes and significantly attenuates YAP-induced cardiac regeneration. Live imaging reveals a distinct mode of sarcomere disassembly driven by enhanced microtubule polymerization, wherein microtubule plus-ends directly interact with α-actinin and displace α-actinin fragments, thereby facilitating sarcomere breakdown. Furthermore, the YAP5SA-S94A mutation, which disrupts the YAP and TEA domain interaction, significantly reduces YAP5SA-induced microtubule growth, sarcomere disassembly, and cell cycle activity. Mechanistically, cleavage under targets and release using nuclease combined with RNA sequencing identified direct YAP targets, including Ajuba and Tuba1b , which are critical for microtubule growth. CM-specific knockdown of Tuba1b attenuates YAP-driven sarcomere disassembly. CONCLUSIONS: These findings identify microtubule networks as an essential regulator modulating CM dedifferentiation and sarcomere reorganization, which is critical for CM cytokinesis and cardiac regenerative repair.