Abstract Body: Infants exposed to diabetic pregnancy are at higher risk of cardiomyopathy at birth and early onset cardiovascular disease (CVD) as adults. We have previously used a rat model to demonstrate that a combination of high fat diet (HFD) and late gestational diabetes causes impaired cardiac function in first generation (F1) newborn and adult offspring via mitochondrial dysfunction and metabolic stress. The objective of this study was to determine whether grandmaternal HFD and diabetes can cause inheritable CVD in second generation offspring (F2), even if the first generation is cross fostered during lactation and fed a standard control diet throughout life. Myocardial function in the F2 newborns was assessed using echocardiography and mitochondrial function in the isolated neonatal cardiomyocytes (NRCM) was assessed using extracellular flux analyses. Exposed F2 newborns had a higher birthweight (p=0.03) and blood glucose (p<0.0001) compared to control newborns and F1 generation parents (p=0.0002 and p<0.0001). Of significant interest, exposed F2 newborns had higher heart rates (p<0.0001), heart:body weight ratios (p<0.0001) and systolic dysfunction which was evident from lower ejection fraction (p<0.0001) and fractional shortening (p<0.0001) compared to control F2 newborns. While control and combination exposed NRCM had similar basal respiration, exposed F2 NRCM had a higher proton leak (p=0.0004) which indicates damage to the inner mitochondrial membrane and/or electron transport chain complex. There was also shift to higher glycolysis at baseline (p=0.005), but blunted glucose-induced glycolytic rate (p=0.04) in F2 newborns compared to controls. Overall results suggest that maternal HFD and diabetes causes multigenerational programming of cardiometabolic dysfunction in F2 offspring despite the F1 parent having normal diet, weight, and euglycemia during the pregnancy.