Background <p>The gut microbiota plays a crucial role in providing essential stimulatory signals for the development of the immune system during the early stages of life. Cesarean delivery is associated with altered vertical microbial transmission and may affect early immune priming. Currently, only a limited number of studies have investigated the interactions among gut microbiota, fecal metabolites, and immune function in neonates delivered by cesarean section, which underscores the necessity for further research.</p> Methods <p>We performed metagenomic sequencing and untargeted metabolomics to compare the gut microbiota and fecal metabolites of neonates born through cesarean delivery (<i>n</i> = 18) and vaginal delivery (<i>n</i> = 20). RNA sequencing (RNA-Seq) was utilized to identify differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs). Immune profiling involved flow cytometry analysis to determine the proportions of Th1 and Th2 cells, ELISA-based quantification of plasma IFN-γ, IL-12p70, IL-4, and IL-10, and assessment of STAT4 and STAT6 expression via ELISA and Western blot. Multi-omics integration was applied to elucidate the systemic impact of cesarean delivery on the neonatal gut microbiome, metabolome, and immune system.</p> Results <p>The composition and functional features of the gut microbiota, and fecal metabolite profile, were significantly altered in Cesarean group. PBMC gene expression also showed marked differences, presenting a Th2-biased immune response and enrichment of genes associated with systemic lupus erythematosus and primary immunodeficiency. Flow cytometry and ELISA confirmed a Th1/Th2 imbalance, while Western blot revealed decreased STAT4 and increased STAT6 expression in the Cesarean group. Multi-omics analysis indicates that <i>Bacteroides sp.</i> is associated with alterations in fecal metabolite in neonates delivered via cesarean section. The reduced abundance of <i>Bacteroides sp.</i> and <i>Bacteroides fragilis</i> correlated with Th1/Th2 dysregulation. Additionally, gut microbiota changes were correlated with variations in the host oxidative phosphorylation pathway via fecal phosphate levels.</p> Conclusions <p>This multi-omics study reveals an association between the mode of delivery and distinct gut microbiota structure, fecal metabolite profiles, and immune development during early life. This provides a framework for investigating the potential connection between early-life immune programming and mode of delivery.</p> Clinical trial number <p>Not applicable.</p>