Abstract Body: Cardiomyocytes (CMs) undergo metabolic and contractile reprogramming during cardiac stress and injury. This reprogramming response includes a fuel metabolic shift to a more fetal-like or immature state with downregulation in fatty acid (FA) oxidation and increased glucose utilization. We sought to test the hypothesis that these fuel shifts trigger a broader CM re-programming. To this end, we targeted the nuclear receptor peroxisome proliferator-activated receptor α (PPARα), a master regulator of cellular FA utilization, in adult mouse CMs in vivo and in cultured neonatal rat ventricular myocytes (NRVMs) and assessed fuel metabolism, structural and functional remodeling, and cell cycle activity. First, PPARα was inducibly targeted in CMs of adult mice (cs-PPARαKO mice) and cardiac fuel utilization shifts, including increased cardiac glucose utilization, were observed through in vivo ¹³C-isotope tracer studies. RNA-sequencing and proteomics of PPARα-deficient CMs revealed a broad program of dedifferentiation indicative of a fetal-like shift in fuel utilization, sarcomeric remodeling and signatures of cell cycle reentry. Isolated mitochondria from PPARα KO hearts showed reduced membrane potential and substrate oxidation, consistent with metabolic immaturity. PPARα KO CMs exhibited sarcomere disorganization and cell rounding by morphological analysis and functional studies showed decreased sarcomere shortening and delayed relaxation, suggesting structural and functional immaturity. PPARα KO CMs showed increased Rb phosphorylation and EdU incorporation, but nucleation and cell number remained unchanged, indicative of renewed cell cycle activity that is unable to progress through G2/M for proliferation. In NRVMs, PPARα siRNA knockdown enhanced cell cycle activity and cytokinesis, induced cytoskeletal remodeling and altered calcium handling, hallmarks of a dedifferentiated state. Interestingly, loss of the gluconeogenic enzyme fructose-bisphosphatase 2 (Fbp2), a CM PPARα target that is robustly downregulated in PPARα KO hearts, promoted many similar structural and functional signatures of dedifferentiation. This implicates a direct mechanistic role of modulating glycolytic flux in CM fetal reprogramming. These findings indicate that reprogramming fuel utilization from FA oxidation to glycolysis, possibly through Fbp2 downregulation, triggers CM dedifferentiation and drives cell cycle reentry through a yet-to-be defined metabolic signaling mechanism.