Abstract Body: Unlike adult mammals, the neonatal mouse has an exceptional regenerative capacity following myocardial injury. Our goal was to decipher and define novel factors and mechanisms that have a role in regeneration of the injured adult mouse heart. FOXK1 is a transcription factor that regulates cell cycle kinetics, myogenic stem cell proliferation, and skeletal muscle regeneration. During development, its expression is restricted to progenitors, somites and the heart. In these studies, we identified a novel role for FOXK1 as a regulator of cardiomyocyte (CM) proliferation and regeneration through the regulation of Gli2. Analysis of single nucleus RNAseq datasets of neonatal hearts revealed that FOXK1 was expressed in neonatal CMs. qPCR analysis from P1 hearts of control and Foxk1 null mice confirmed this finding and demonstrated dysregulation of the cell cycle regulators, Ccne1 and p21, in the absence of Foxk1. Control and Foxk1 null mice were pulsed with EdU from P3 to P7 which demonstrated that the absence of Foxk1 led to a decrease in CM proliferation in-vivo. Then, using a transgenic mouse model that we engineered to overexpress FOXK1 in muscle (MCK-Foxk1), we observed CM proliferation in adult MCK-Foxk1 mice, which showed increased incorporation of EdU and increased pH3 expression compared to control. Bulk RNA-seq analysis of P1, P7, and P14 hearts indicated increased CM proliferation in the MCK-Foxk1 mouse and revealed Gli2 as a potential downstream target of FOXK1. A gel shift assay was performed, which demonstrated that FOXK1 was able to bind to a FOXK1 motif upstream of the Gli2 gene. ChIP-PCR further verified that Gli2 was a direct downstream target of FOXK1 in the embryonic stem cell-embryoid body (ES-EB) system. We then performed ligation of the left anterior descending coronary artery (LAD) in adult control and MCK-Foxk1 mice. Echocardiography following the LAD-ligation injuries showed improved cardiac function in MCK-Foxk1 transgenic mice compared with controls. These results identify FOXK1 as a regulator of CM proliferation and heart regeneration and suggest that this function is mediated through the regulation of Gli2 gene expression.