Abstract Body: We reported that Krüppel-like factor 9 (Klf9) regulates metabolic adaptations in neonatal cardiomyocytes. In this study, we characterize conditional Klf9 knock-in (Klf9KI) mice with 3-4-fold increase in Klf9 expression when crossed with αMHC-Cre. Our data shows that Klf9KI mice develop spontaneous hypertrophy by 4wks, followed by cardiac dysfunction and failure by 8wks (EF=24 ±1%; FS=11±0.5%), and early mortality by 12-14 wks. Transcriptome analysis in 1wk, 4wk and 8wks old hearts shows that the number of differentially expressed genes increases over time, with significant changes observed in 108 genes at 1 wk, and further dysregulation of 949 genes and 1247 genes at 4wks and 8wks, respectively. Consistent with Klf9's predicted role as a transcriptional repressor, 64.81% of differentially expressed genes are downregulated at 1wk. Functional annotation reveals a dysregulation of mostly metabolic genes including those involved in lipid metabolism (Bdh1), glutathione pathways (Gstk1), and carbohydrate metabolism (Fbp2). By 4wks and 8wks, along with dysregulation of metabolic pathways, we observe upregulation of genes involved in apoptosis (Bcl2), innate immunity (Tlr genes), and extracellular matrix remodeling (Col8a1). Untargeted metabolomics at 4wks identifies significant alterations in 125 tissue metabolites [VIP >1.5, ILog2FCI ≥0.5, p<0.05]). Pathway enrichment analysis shows Arachidonic acid, Lipid and Fatty acids metabolism and biosynthesis, Sucrose and Glutathione metabolism as top enriched metabolite sets. These findings correlate with dysregulated genes identified in 4wks old Klf9KI mice (RNA-seq). To assess mitochondrial function, we measured mitochondrial electron transport chain complex activities in Klf9KI and Wt-Cre mice at 2 and 4wks. Basal mitochondrial respiration decreases at both time points, while complex II (succinate) and IV (ascorbate +TMPD) activities decline significantly by 4wks. A 3.5-fold increase in ROS production is observed in these hearts, consistent with a decline in mitochondrial function. In conclusion, constitutive expression of Klf9 disrupts transcriptional and metabolic homeostasis, which precipitates spontaneous progressive hypertrophy, dysfunction, and early onset of heart failure.