Nutritional constraints during gestation result in persistent phenotypic changes to the offspring which are associated with increased risk of chronic non-communicable disease (Bateson et al. 2004; Gluckman & Hanson 2004). The molecular mechanism(s) underlying this process is not known. We investigated the effect of reduced maternal protein intake during pregnancy on the regulation of hepatic transcription factors that control lipid metabolism in the offspring. Female Wistar rats (n = 5 / group) were fed diets containing 18%(w/w) or 9%(w/w) protein (both with 1mg/kg folic acid), or 9%(w/w) protein + 6mg/kg folic acid (9%F) from conception until delivery, and chow throughout lactation. Pups were weaned at 28 days and humanely killed at 34 days. One liver was analysed per litter. The methylation status of the peroxisomal proliferator activated receptor (PPAR) α and γ promoters was determined by real time PCR after digestion with Aci1 (Pham et al. 2003). PPARα, γ and acyl-CoA oxidase (AOX), a PPARα target gene, mRNA expression was determined by RTPCR (Burdge et al. 2004). The 9% protein diet decreased hepatic PPARα promoter methylation (33.7%, p<0.001) and increased PPARα mRNA expression (854%, p<0.0001) in the offspring compared to controls. AOX expression was increased (317%, p<0.001) in the 9% group. There were no significant differences between the 18% group and the 9%F group. The methylation status of PPARγ1 and PPARγ mRNA expression did not differ between groups. These results show that maternal protein restriction during pregnancy altered the regulation of PPARα expression by decreasing the methylation of promoter sequence. The increase in AOX expression is consistent with up-regulation of PPARα activity, and suggests greater peroxisomal β-oxidation and dysregulation of fatty acid metabolism. The absence of changes to PPARγ expression suggests the effects of prenatal under-nutrition are gene-specific. Prevention of these effects by folic acid shows that altered 1-carbon metabolism is central to the phenotypic changes associated with the restricted diet. These observations provide one causal mechanism linking maternal nutrition during pregnancy, regulation of gene expression and altered phenotype in the offspring.