In this paper, the anti-aging effect of polysaccharides from Athyrium multidentatum (Doll.) Ching (AMC) and the underlying mechanisms was investigated exploying D-galactose (D-gal) induced aging mouse model. Cytokine levels were detected by enzyme-linked immunosorbent assay (ELISA). Antioxidant enzyme activity and lipid peroxide content were determined by ultraviolet-visible spectrophotometry. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot assays were performed to assess the genes and proteins expression. Our results suggested that AMC polysaccharides strikingly ameliorated the pathological changes in hippocampus, adjusted the organ indices, increased the serum interleukin-2 (IL-2) content, and decreased the tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) levels. Furthermore, AMC polysaccharides notably augmented superoxide dismutase (SOD)/catalase (CAT) activities and silent information regulator 1 (SIRT1) gene expression, and lowered p53/p21 mRNA and p53 protein levels. Available data obtained with in vivo model suggested that AMC polysaccharides did display marked anti-aging activity. The mechanisms might attribute to their capacities of augmenting antioxidant enzyme activities, improving immunity function, suppressing lipid peroxidation reaction and regulating SIRT1/p53/p21 signaling pathway.