Myocardial infarction (MI) significantly compromises the integrity of the cardiac microvascular endothelial barrier, leading to enhanced leakage and inflammation that contribute to the progression of heart failure. While HIF2α is highly expressed in cardiac endothelial cells (ECs) under hypoxic conditions, its role in regulating microvascular endothelial barrier function during MI is not well understood. In this study, we utilized mice with a cardiac-specific deletion of HIF2α, generated through an inducible Cre (Cdh5Cre-ERT2) recombinase system. These mice exhibited no apparent phenotype under normal conditions. However, following left anterior descending (LAD) artery ligation-induced MI, they showed increased mortality associated with enhanced cardiac vascular leakage, inflammation, worsened cardiac function, and exacerbated heart remodeling. These outcomes suggest a protective role for endothelial HIF2α in response to cardiac ischemia. Parallel investigations in human cardiac microvascular endothelial cells (CMVECs) revealed that loss of ecHif2α led to diminished endothelial barrier function, characterized by reduced tight-junction protein levels and increased cell death, along with elevated expression of IL6 and other inflammatory markers. These effects were substantially reversed by overexpressing ARNT, a critical dimerization partner for HIF2α during hypoxia. Additionally, ARNT deletion also led to increased CMVEC permeability. Interestingly, ARNT, rather than HIF2α itself, directly binds to the IL6 promoter to suppress IL6 expression. Our findings demonstrate the critical role of endothelial HIF2α in response to MI and identify the HIF2α/ARNT axis as a transcriptional repressor, offering novel insights for developing therapeutic strategies against heart failure following MI.