Exposure to a high-fat (HF) diet in utero is associated with increased incidence of cardiovascular disease, diabetes, and metabolic syndrome later in life. However, the molecular basis of this enhanced susceptibility for metabolic disease is poorly understood. Gene expression microarray and genome-wide DNA methylation analyses of mouse liver revealed that exposure to a maternal HF milieu activated genes of immune response, inflammation, and hepatic dysfunction. DNA methylation analysis revealed 3360 differentially methylated loci, most of which (76%) were hypermethylated and distributed preferentially to hotspots on chromosomes 4 [atherosclerosis susceptibility quantitative trait loci (QTLs) 1] and 18 (insulin-dependent susceptibility QTLs 21). Interestingly, we found six differentially methylated genes within these hotspot QTLs associated with metabolic disease that maintain altered gene expression into adulthood (Arhgef19, Epha2, Zbtb17/Miz-1, Camta1 downregulated; and Ccdc11 and Txnl4a upregulated). Most of the hypermethylated genes in these hotspots are associated with cardiovascular system development and function. There were 140 differentially methylated genes that showed a 1.5-fold increase or decrease in messenger RNA levels. Many of these genes play a role in cell signaling pathways associated with metabolic disease. Of these, metalloproteinase 9, whose dysregulation plays a key role in diabetes, obesity, and cardiovascular disease, was upregulated 1.75-fold and hypermethylated in the gene body. In summary, exposure to a maternal HF diet causes DNA hypermethylation, which is associated with long-term gene expression changes in the liver of exposed offspring, potentially contributing to programmed development of metabolic disease later in life.