Polypeptides are important active substances in Agaricus blazei. To investigate the anti-aging effect of Agaricus blazei polypeptide (ABp), ABp was prepared by hydrolyzing Agaricus blazei protein with pepsin, and purified by column chromatography using a protein purification system. A mouse aging model was established by injecting D-galactose. The effects of ABp on aging model mice were investigated by behavioral experiments, serum biochemical indicators and transcriptome sequencing. Furthermore, Western blotting was used to explore the potential mechanism of ABp. The results indicated that the molecular weight of ABp was 3.3 kDa after separation and purification. ABp could significantly inhibit the elevated levels of reactive oxygen species and malondialdehyde in mouse serum caused by D-galactose, significantly enhanced the total antioxidant capacity and catalase activity in mouse serum. The transcriptome sequencing results showed significant differences in the expression levels of 295 genes in mouse hippocampus between ABp group and model group. Compared with the aging model group, ABp upregulated 79 genes and downregulated 216 genes in mouse hippocampus. The results of Western blotting indicated that the potential mechanism of ABp anti-aging might be related to the Keap1/Nrf2/P53 signaling pathway. This study aims to provide a theoretical basis for the development of anti-aging functional food of Agaricus blazei.