We investigated the anti-aging effects of velvet antler polypeptide on D-galactose (D-gal)-induced aging mice. D-gal-induced aging mice were established and randomly divided into five groups, the control, model, vitamin E (VE), velvet antler polypeptide low-dose and velvet antler polypeptide high-dose groups. The Morris water maze test was used to evaluate the learning and memory abilities of aging mice. Hippocampal neurons were observed via hematoxylin-eosin staining and transmission electron microscopy. Biochemical methods were used to detect the activities of superoxide dismutase, malonaldehyde and other enzymes and evaluate the influence of velvet antler polypeptide on the antioxidant capacity of aging mice. Using 16S rRNA gene sequencing and meristem technology, we assessed the effect of velvet antler polypeptide on aging mice’s intestinal flora and fatty acid metabolism. The experimental results showed that velvet antler polypeptide could significantly improve aging mice’s learning and cognitive abilities, increase the activities of superoxide dismutase, glutathione peroxidase, and catalase in the serum decrease the malonaldehyde content. Intestinal microecological analysis showed that velvet antler polypeptide could significantly increase the beneficial bacterial genus Lactobacillus abundance. Western blot analysis further demonstrated that velvet antler polypeptide could promote fatty acid metabolism by activating peroxisome proliferator-activated receptor \alpha (PPAR\alpha) and upregulating the expression of the downstream enzymes carnitine-palmitoyl transferase-1 A and acyl-CoA oxidase 1 while downregulating that of apolipoprotein E4 (APOE4), thereby reducing fatty acid accumulation and increasing adenosine-triphosphate (ATP) production. Therefore, velvet antler polypeptide improves the intestinal microecology and activates the PPAR\alpha/APOE4 pathway to regulate fatty acid metabolism.