Many commercial human infant formulas contain a protein content greater than what is naturally found in human breast milk. Prolonged consumption of protein greater than nutritional requirements in early infancy has been linked to increased risk of obesity and type 2 diabetes, which is characterized by decreased insulin sensitivity and impaired glucose homeostasis. Due to the negative impacts of low-protein diets on infant growth, their use in commercial formula is limited. Alternatively, dietary restriction of insulinogenic amino acids (IAA, i.e., Leu, Ile, Val, Thr, Phe, Arg, and Ala) may be considered. This study aimed to determine the impact of IAA restriction in formula on glucose and lipid metabolism in a neonatal piglet model for human infants. 32 seven-day-old Yorkshire barrows were randomly assigned to one of three dietary treatment groups for 21 days: 1) NR: 0% IAA restriction; 2) R50: 50% IAA restriction; and 3) R75: 75% IAA restriction; with each diet being isonitrogenous and isocaloric. After 21 days, all animals were sacrificed, and liver, skeletal muscle, and white adipose tissue (WAT) samples were collected. Using RT-qPCR the expression of glucose and lipid metabolism and insulin signaling genes in target tissues was determined. Data were analyzed with Univariate GLM with Dunnett’s post-hoc (SPSS®). Relative to NR, the R75 treatment group increased the mRNA abundance of key rate-limiting glycolytic enzymes and glucose transporters including hepatic glucose transport 1 (GLUT 1), hepatic pyruvate kinase (PFKL), hepatic pyruvate kinase liver type (PKLR), and WAT glucokinase (GCK). R50 increased the mRNA expression of hepatic and muscular PKLR. Insulin signaling markers significantly improved via increased mRNA of muscular serine/threonine kinase 1 (AKT) and insulin receptor substrate 1 (IRS1) when R75 was compared to NR. IAA restriction by 75% increased the mRNA expression of hepatic fibroblast growth factor 21 (FGF-21), with R50 increasing the mRNA expression of hydroxyacyl-CoA dehydrogenase (HADH). In conclusion, restriction of dietary IAA improved glucose and lipid metabolism in a neonatal piglet model likely through increasing the rates of glycolysis via upregulation of key glycolytic pathway enzymes, and gene expression of key enzymes involved in lipid metabolism.