Maternal undernutrition around the time of conception is associated with an increased risk of insulin resistance in adulthood. We determined the effect of maternal undernutrition in the periconceptional period (PCUN, i.e., 60 days prior to 6 days after conception) and the preimplantation period (PIUN, i.e., 0-6 days after conception) on mRNA expression and protein abundance of key insulin-signaling molecules as well as the global microRNA expression in quadriceps muscle of singleton and twin fetal sheep in late gestation. In singleton fetuses, exposure to PCUN resulted in lower protein abundance of PIK3CB (P < 0.01), PRKCZ (P < 0.05), and pPRKCZ (Thr410) (P < 0.05) in skeletal muscle compared to controls. In PIUN singletons, there was a higher protein abundance of IRS1 (P < 0.05), PDPK1 (P < 0.05), and SLC2A4 (P < 0.05) compared to controls. In twins, PCUN resulted in higher protein abundance of IRS1 (P < 0.05), AKT2 (P < 0.05), PDPK1 (P < 0.05), and PRKCZ (P < 0.001), while PIUN also resulted in higher protein abundance of IRS1 (P < 0.05), PRKCZ (P < 0.001), and SLC2A4 (P < 0.05) in fetal muscle compared to controls. There were specific patterns of the types and direction of changes in the expression of 22 microRNAs in skeletal muscle after exposure to PCUN or PIUN and clear differences in these patterns between singleton and twin pregnancies. These findings provide evidence that maternal undernutrition around the time of conception induces changes in the expression of microRNAs, which may play a role in altering the abundance of the key insulin-signaling molecules in skeletal muscle and in the association between PCUN undernutrition and insulin resistance in adult life.