Iron is a crucial micronutrient, and its deficiency can have detrimental effects on the health of infants. Dietary polysaccharide-iron (III) complexes (PICs) are promising for addressing iron deficiency due to their minimal adverse reactions and high iron absorption rate. This study aimed to investigate the effects of dietary Enteromorpha prolifera polysaccharide-Fe (III) complex (EP-Fe) on newborns, using 3-day weaned piglets as the iron-deficiency model. Results showed that EP-Fe improved iron levels and promoted intestinal development in piglets. Transcriptome sequencing revealed that EP-Fe increased the survival of intestinal epithelial cells under hypoxia by upregulating the expression of genes that promote the development of the vascular system. Additionally, EP-Fe enhanced the mucosal barrier functions by inhibiting myosin light chain kinase (MLCK)/phosphorylated myosin light chain (p-MLC) signaling pathway to increase the expression of intestinal tight junction proteins. Furthermore, the 16S rRNA gene sequencing of gut microbiota showed that EP-Fe promoted the enrichment of Bacteroides_fragilis and other gut microbes that can metabolize carbohydrates. In conclusion, EP-Fe is an effective iron supplement for newborns, and it can be developed as a comprehensive nutritional supplement.