Vitamin A deficiency is one of the most common dietary deficiencies in the developing world and is a major health concern where it is associated with increased risk of fetal and infant mortality and morbidity. Early studies in the rat demonstrated that, in addition to respiratory problems, neonates showed evidence of mobility problems in response to moderate vitamin A deficiency. This study investigated whether moderate deficiency of this vitamin plays a role in regulating key skeletal muscle regulatory pathways during development. 30 female rats were fed vitamin A moderate (VAM) or vitamin A sufficient (VAS) diets from weaning and throughout pregnancy. Fetal and neonatal hindlimb and muscle samples were collected on days 13.5, 15.5, 17.5 and 19.5 of pregnancy and one day following birth. Mothers fed the VAM diet had reduced retinol concentrations at all time points studied (P<0.01) and neonates had reduced relative lung weights (P<0.01). Fetal weight and survival did not differ between groups but neonatal survival was lower in the VAM group where neonates had increased relative heart weights (P<0.05). Analysis of myogenic regulatory factor expression and calcineurin signaling in fetuses and neonates demonstrated decreased protein levels of myf5 (50% at 17.5dg (P<0.05)), myogenin (70% at birth (P<0.001)) and Myosin Heavy Chain-fast (50% at birth (P<0.05) in response to moderate vitamin A deficiency. Overall, these changes suggest that Vitamin A status during pregnancy may have important implications for fetal muscle development and subsequent muscle function in the offspring.