Deer antler has been a staple food for millennia, yet its precise immunomodulatory mechanisms remain unexplored. In our study, we systematically investigated the effects and mechanisms of deer antler water extract (DAWE) on cyclophosphamide-induced immunosuppressive mice. We identified various bioactive components in DAWE, including monosaccharides, nucleosides, phospholipids, amino acids, lipids, and organic acids. Following 14 days of administration of DAWE (260 mg/kg) significantly enhanced immunity in immunosuppressed mice, alleviating spleen injury, boosting immune cell proliferation, and increasing immunoactive substances production. Moreover, DAWE effectively mitigated intestinal barrier damage by promoting goblet cell and IgA-secreting cell proliferation and upregulating the expression of occludin-1 and claudin-1. Additionally, DAWE modulated gut microbiota diversity, composition, and metabolic activity. The metabonomic analysis highlighted the pivotal role of glutamine and glutamate metabolic pathways in DAWE's immune regulatory effects. These findings provide valuable insights into DAWE as a potential immunomodulator and functional food, warranting further exploration in clinical applications.