Maternal undernutrition around the time of conception resulted in insulin resistance and glucose intolerance in adulthood. To determine the programming effect of maternal undernutrition around the time of conception on the hepatic insulin signalling and gluconeogenic factors, we quantified the mRNA and protein abundance of insulin signalling and gluconeogenic factors using qRT-PCR and Western blotting, respectively, and global microRNA expression was quantified using deep sequencing methodology in the liver of late gestation sheep fetuses exposed to periconceptional (PCUN: -60d-7d) or preimplantation (PIUN: 0d-7d) undernutrition compared to controls. We found that PEPCK-C mRNA and protein abundance of PEPCK-C, IRS-1, p110β, PTEN, CREB, pCREB(Ser133) was decreased in singletons exposed to PCUN and PIUN. Interestingly, in twins, IRS-1, p85, p110β, PTEN, Akt2, pAkt(Ser473) and pFOXO-1(Thr24) protein abundance was increased, but there was a decrease in PEPCK-C mRNA and a paradoxical increase in PEPCK-C protein in PCUN and PIUN groups. We also found that PCUN or PIUN alters the expression of 23 microRNAs including miR-130a, miR-19a, miR-19b, miR-335, miR-34c, miR-369, miR-379, miR-106b and let-7a. These findings show that undernutrition during the first week of embryonic development is sufficient to program a change in the abundance of insulin signalling and gluconeogenic factors, which may be mediated by altered expression of specific miRNAs, and the effect is different in singletons and twins.