Polysaccharides extracted from Agrocybe aegerita (AAPS) have various physiological effects. In this study, we used the naturally aging Drosophila melanogaster and D-galactose-induced aging mice as animal models to study the anti-aging effects of AAPS via the alleviation of oxidative stress and regulation of gut microbiota. Results showed that AAPS could significantly prolong lifespan and alleviate oxidative stress induced by H₂O₂ of Drosophila melanogaster. In addition, AAPS significantly increased the activities of antioxidant enzymes in Drosophila melanogaster and mice, and reduced the content of MDA. Furthermore, AAPS reshaped the disordered intestinal flora, increased the abundance ratio of Firmicutes to Bacteroidetes, and increased the abundance of beneficial bacteria Lactobacillus. Our results demonstrated that AAPS had good antioxidant and potential anti-aging effects in vivo.