Summary Maternal diabetes induces congenial heart defects (CHDs) formation and the underlying mechanism is still unclear. Maternal diabetes induces hypoxia in the developing embryo and short-term gestational hypoxia induces CHDs. Hypoxia and DNA hypermethylation have been interlinked in human diseases. DNA hypermethylation is implicated in CHDs including hypoplastic left heart syndrome (HLHS), a complex and severe CHD type. We found that maternal diabetes enhanced hypoxia and increased DNA methylation in the developing heart. Hypoxia inducible factor 1 alpha (HIF-1α) up-regulated the two de novo DNA methyltransferase (DNMT3a and DNMT3b) in cardiac progenitors in the developing mouse hearts or derived from human inducible pluripotent stem cells (iPSCs). Blockage of DNA hypermethylation by removing DNMT3a and DNMT3b in early cardiac Nkx2.5+ progenitors ameliorated all CHD types in diabetic pregnancy. Thus, we hypothesize that maternal diabetes induces hypoxia and triggers the activation of the hypoxia inducible factor 1 alpha (HIF-1α) pathway, which induces DNA hypermethylation by up-regulating DNMT3a/b. Inhibition of hypoxia, HIF-1α or DNA hypermethylation or double DNMT3a/b deletion abrogates the functional deficits in cardiac progenitors leading to CHD reduction and improvement of cardiomyocyte and cardiac function. First heart field defects contribute to HLHS formation and cardiac dysfunction in this severe type of CHDs. To test our hypothesis, we proposed three specific aims. Aim 1 will determine whether maternal diabetes-induced hypoxia is responsible for DNA hypermethylation in early cardiac progenitors leading to CHD formation. We will examine whether hypoxia increases DNA methylation in early cardiac progenitors by up-regulating DNMT3a/b expression leading to CHDs in diabetic pregnancy. Aim 2 will investigate the role of maternal diabetes-induced DNA hypermethylation in gene dysregulation that results in functional defects in early cardiac progenitors and the first heart field. We will determine whether DNA hypermethylation in both heart fields alters gene expression leading to CHDs and cardiomyocyte dysfunction in diabetic pregnancy by using DNMT3a/b double deletion in early cardiac progenitors. Aim 3 will determine whether heightened HIF-1α activity and consequent DNA hypermethylation contribute to cardiomyocyte dysfunction of HLHS in diabetic pregnancy. We hypothesize that persistent activation of the HIF-1α pathway and DNA hypermethylation contribute to cardiomyocyte dysfunction in maternal diabetes-induced CHDs. Successful completion will dissect the critical role of hypoxia and DNA methylation in diabetes-induced CHDs and provide mechanistic insights for improving cardiomyocyte function in CHD patients.