We have investigated the effects of embryo number and maternal undernutrition imposed either around the time of conception or before implantation on hepatic lipid metabolism in the sheep fetus. We have demonstrated that periconceptional undernutrition and preimplantation undernutrition each resulted in decreased hepatic fatty acid β-oxidation regulators, PGC1α (P<0.05), PDK-2 (P<0.01) and PDK-4 (P<0.01) mRNA expression in singleton and twin fetuses at 135-138 days gestation. In singletons, there was also lower hepatic PDK-4 (P<0.01), CPT-1 (P<0.01), and PKC (P<0.01) protein abundance in the PCUN and PIUN groups, and a lower protein abundance of PDPK-1 (P<0.05) in the PCUN group. Interestingly in twins, the hepatic protein abundance of pAMPK (Ser485) (P<0.01), pPDPK-1 (Ser 41) (P<0.05) and PKC (P<0.05) was higher in the PCUN and PIUN groups, and hepatic PDK-4 (P<0.001) and CPT-1 (P<0.05) protein abundance was also higher in the PIUN twin fetus. We also found that the expression of a number of microRNAs were altered in response to PCUN or PIUN and that there is evidence that these changes may underlie the changes in the protein abundance of key regulators of hepatic fatty acid β-oxidation in the PCUN and PIUN groups. Therefore, embryo number and the timing of maternal undernutrition in early pregnancy have a differential impact on hepatic microRNA expression and on the factors that regulate hepatic fatty acid oxidation and lipid synthesis.