AbstractPericonceptional diet may persistently influence DNA methylation levels with phenotypic consequences. However, a comprehensive assessment of the characteristics of prenatal malnutrition-associated differentially methylated regions (P-DMRs) is lacking in humans. Here we report on a genome-scale analysis of differential DNA methylation in whole blood after periconceptional exposure to famine during the Dutch Hunger Winter. We show that P-DMRs preferentially occur at regulatory regions, are characterized by intermediate levels of DNA methylation and map to genes enriched for differential expression during early development. Validation and further exploratory analysis of six P-DMRs highlight the critical role of gestational timing. Interestingly, differential methylation of the P-DMRs extends along pathways related to growth and metabolism. P-DMRs located in INSR and CPT1A have enhancer activity in vitro and differential methylation is associated with birth weight and serum LDL cholesterol. Epigenetic modulation of pathways by prenatal malnutrition may promote an adverse metabolic phenotype in later life.