ABSTRACT: Few studies focused on the effects of iron on characterizing alterations of metabolic processes in neonatal piglets. In the present study, 16 neonatal piglets were randomly assigned to two groups. In the first group piglets were given an intramuscularly injection of iron dextran at 150 mg as a positive control (CON) and the second group were not supplemented with iron as a negative control for iron deficiency (ID). At day 8, iron status, serum biochemical parameters, serum metabolome, hepatic histology, and hepatic expression of genes for the metabolism were analyzed. Results indicated that piglets without iron supplementation had significantly reduced iron values and increased blood urea nitrogen concentrations at day 8 (p < 0.05). Analysis of serum metabolome revealed that concentrations of serum lysine, leucine, tyrosine, methionine, and cholesterol were significantly decreased while concentrations of 3-Methyldioxyindole, chenodeoxycholate acid, indoleacetic acid, icosadienoic acid, phenylpyruvic acid, pantothenic acid, ursocholic acid, and cholic acid were significantly increased in iron deficient piglets (p < 0.05). Furthermore, expressions of cyp7a1 and the urea cycle enzyme (ornithinetranscarbamoylase and argininosuccinate synthetase) were significantly increased in iron deficient pigs (p < 0.05). The present experimental results indicated that neonatal piglets without iron supplementation drop to borderline anemia within 8 days after birth. Iron deficiency led to a series of metabolic changes involved in tyrosine metabolism, phenylalanine metabolism, bile secretion, primary bile acid biosynthesis, steroid biosynthesis, and upregulated activities of the urea cycle enzymes in the liver of neonatal piglets, suggesting early effects on metabolic health of neonatal piglets.