Abstract Body: Krüppel-like factors (Klfs) regulate cellular processes, including metabolism, differentiation, and proliferation, which have implications in development and diseases of the heart. We reported Klf9’s role in transcriptional control of metabolic genes and the GR-Klf9 axis in metabolic adaptation in response to Dexamethasone in neonatal myocytes. Here, we examine the role of Klf9 in adult hearts undergoing pressure overload-induced hypertrophy. Our RNA polymerase II (pol II) ChIPSeq data from mouse hearts undergoing transverse aortic constriction (TAC) induced hypertrophy show reduced pol II occupancy across the Klf9 gene compared to sham hearts, suggesting reduced transcription. Consistently, we observe reduced Klf9 transcript and protein levels in TAC hearts compared to sham, indicating decreased Klf9 expression. Klf9-ChIPSeq identified 5238 genes with Klf9 binding, with 2250 with MaxTag ≥50. 51% of the genes showed decreased Klf9 genomic binding in TAC vs Sham hearts. KEGG pathway showed metabolic pathways on top of the list with 131 genes, including those involved in carbon metabolism, insulin signaling, fatty acid degradation, glycolysis/gluconeogenesis, amino acid, and nucleotide metabolism. Conversely, 22% of genes showed increased Klf9 binding, mostly those involved in innate immunity, apoptosis, mitochondrial organization, and autophagy. Further, we subjected inducible conditional Klf9 knock-in (Klf9KI) mice to sham/TAC operations for 2 weeks, followed by functional and molecular analysis. Contrary to our hypothesis, restoring Klf9 levels with TAC showed systolic dysfunction in Klf9KI-TAC (%EF-38.65±1.9,%FS-18.34±0.77) compared to Wt-TAC (%EF-53.88±3.33,%FS-27.03±1.96) hearts, with no significant change in LV mass within the TAC groups (75.66±13.73 vs. 78.7±8.71). Interestingly, our preliminary data examining gene expression showed a significant increase in hypertrophy markers, Mhy7 (bMHC) and NPPB (BNP), and a decrease in Hmgcs2 (Klf9 target from ChIPSeq) in Klf9KI mice within sham and TAC groups. We conclude that Klf9 expression is critical for metabolic homeostasis, and dysregulated Klf9 levels are sufficient for transcriptional changes and with stress can precipitate early cardiac dysfunction and failure.