Early-life nutrition significantly impacts neonatal health and development, where nutrient deficiencies or excesses can lead to various health complications. This is particularly true for iron nutrition. Exclusively breast-fed infants are susceptible to iron deficiency, prompting the use of iron-fortified diets. However, excessive iron exposure poses risks, including disruption of the gut microbiome. We investigated the complex interactions between dietary iron levels, gut microbiota, and specific dietary interventions in neonatal and pathogenic Escherichia coli (E. coli)-challenged weaned pigs, offering insights applicable to both animal and human health and nutrition.Using a piglet model as a translational model for human infants, we explored the impact of high iron fortification and inulin or synbiotic supplementation on iron homeostasis and trace mineral bioavailability to better understand the biological functions of iron, the regulation of iron homeostasis, and the health consequences of iron imbalance in early infancy. Twenty-four piglets were randomly assigned to four treatments, receiving either iron-adequate milk (AI), high-iron milk (HI), HI milk with 5% inulin (HIP), or HIP milk with an oral gavage of Ligilactobacillus agilis YZ050 (HIS). The study revealed that HI fortification increased hemoglobin (Hb), hematocrit (Hct), and serum iron levels but also caused hepatic tissue iron deposition and concurrently reduced zinc (Zn) and copper (Cu) absorption. Inulin supplementation in HIP and HIS diets attenuated hepatic iron overload and decreased colonic and fecal iron levels, suggesting that inulin has the potential to modulate systemic iron utilization and mitigate some of the adverse effects of excessive iron fortification. Further analysis focused on the impact of iron fortification and inulin supplementation, with or without Ligilactobacillus agilis YZ050 (L. agilis YZ050), on the gut microbiome of piglets. Using 16S rRNA sequencing, the study found that both dietary iron and inulin significantly influenced colonic and fecal microbiota diversity, with inulin exerting a more pronounced effect than iron. Inulin supplementation led to increased relative abundances of beneficial bacterial families such as Lachnospiraceae and Prevotellaceae, while decreasing Bacteroidaceae and Ruminococcaceae. The addition of L. agilis YZ050 did not produce significant changes beyond those caused by inulin alone. These results suggest that inulin supplementation plays a critical role in modulating gut microbiota composition, potentially through inulin fermentation and cross-feeding activities among gut bacteria, thereby mitigating the adverse effects of high iron intake on gut health. To further investigate the implications of dietary iron on health, we examined how host iron status and dietary iron supplementation impact disease resilience and susceptibility to intestinal infection and gut microbiota composition using a weanling pig model with enterotoxigenic E. coli (ETEC) as an infection model. Thirty-two pigs were assigned to four treatments: an adequate iron diet without ETEC challenge (CON), low iron diet (LOI), adequate iron diet (COI), or high iron diet (HII) with ETEC challenge. The study found that LOI pigs experienced more days with diarrhea and had lower body weight gain compared to the other groups, indicating that iron deficiency potentially exacerbates disease severity. HII diet was associated with higher Hb and Hct levels. Furthermore, the gut microbiota analysis revealed distinct shifts in microbial composition associated with dietary iron levels and ETEC infection. These shifts included increased abundance of Peptostreptococcaceae in CON and HII pigs, and higher levels of Veillonellaceae and Megasphaera in LOI pigs, suggesting that dietary iron influences gut microbiota. In conclusion, this dissertation contributes to understanding the intricate relationships between dietary components such as iron and inulin, gut microbiota, and health outcomes in neonatal and weaned pigs. The use of a piglet model provides valuable insights into the physiological and microbiological impacts of dietary interventions, offering a translational approach to improving infant nutrition and managing iron-related health issues. The findings highlight the importance of balancing iron intake to prevent both deficiency and overload, while highlighting the potential benefits of inulin supplementation in mitigating some of these adverse effects by affecting iron utilization and modulating gut microbiota composition. Additionally, maintaining adequate iron status is crucial for reducing susceptibility to ETEC infection and promoting better health outcomes. These insights inform evidence-based strategies for dietary iron optimization in both animal and human contexts.