Increasing diabetes prevalence and life expectancy are two key factors raising the health care burden from the consequences of cardiovascular disease (CVD). Whereas an obesogenic diet in adulthood is a known risk factor for type 2 diabetes and CVD, there is growing evidence that obesogenic diets during pregnancy increase CVD and diabetes susceptibility in the offspring later in life. While most studies focused on changes in vascular physiology that result from maternal high fat (HF) feeding, the metabolic and molecular changes that occur in the offspring’s heart itself remains to be elucidated. The mitochondria play a key role in the normal functioning of the heart, and in the pathogenesis and development of various types of heart disease. We therefore examined whether mitochondrial electron transport chain (ETC) activity and expression of genes with key roles in mitochondrial metabolism were altered in heart tissues of offspring from obese mothers fed a HF diet. Female C57/BL6J mice were maintained under controlled conditions and fed either a HF diet (HF; 45% kcal fat) or standard chow diet (C; 21% kcal fat) 4-6 weeks prior to and during gestation and lactation. Weaned offspring were fed the HF or C diet, generating the dam-offspring groups: C/C, C/HF, HF/C, HF/HF. Hearts were taken from 15-week old male offspring (n=4-5 per offspring group). The left ventricle (LV) was dissected and processed for the analysis of mitochondrial Complex I and II enzyme activity using spectrophotometric assay, and for qPCR of the mitochondrial sirtuin Sirt3, uncoupling proteins UCP2 and UCP3, the adenine nucleotide translocases ANT1 and ANT2, the transcriptional coactivator PGC1α, and the nuclear respiratory factor NRF1. Complex I and II activity was reduced by 1.5 fold (p<0.001, ANOVA) in the HF/HF offspring heart vs C/C. Complex II activity also reduced by 3-fold (p<0.0001) in the HF/C group vs C/C. Sirt3 mRNA level was 2.6-fold lower (p<0.01) in offspring hearts from HF-fed dams (HF/C and HF/HF groups) vs C/C group. UCP2 and UCP3 mRNA levels were 2.3 and 4-fold higher (both at p<0.0001), in HF/HF vs C/C hearts, with UCP2 also increased 5-fold (p<0.0001) in the HF/C hearts vs C/C. Furthermore, ANT1 and ANT2 transcript levels were reduced by more than 1.3-fold (p<0.01) in HF/HF hearts vs C/C. PGC1α mRNA levels were 1.4-fold lower (p<0.01) while NRF1 mRNA levels were 3-fold higher (p<0.0001) in HF/HF hearts vs C/C. NRF1 transcript levels were also 2.5-fold (p<0.0001) higher in the HF/C tissues vs C/C. The results suggest maternal high fat diet during pregnancy and lactation alters mitochondrial ETC activities and expression of genes involved in mitochondrial function and biogenesis. This priming effect in early life increases offspring risk to cardiac pathologies in later life.