The aim of the present study is to characterize the structure of a novel natural polysaccharide from Agrocybe aegirita (AAPS) and evaluate its anti-aging activity. The MALLS and GC–MS analysis indicated that the AAPS with molecular weights of 1.81 × 10 Da was mainly composed by rhamnose, fucose, mannose, and glucose in a molar ratio of 2.90:10.25:3.70:38.27. The FT-IR and NMR analysis showed that the backbone of AAPS was α-L-Rhap-(1→6)-β-D-Glcp-(1→2)-α-L-Fucp-(1→6)-α-D-Glcp-(1→5)-α-L-Araf-(1→4)-β-D-GlcpA-(1→5)-α-L-Araf-(1→6)-α-D-Manp-(1→6)-α-D-Manp-(1→2)-α-L-Fucp-(1→6)-β-D-Glap-(1→2)-α-L-Rhap-(1→6)-β-D-Galp-(1→, which linked with two side chains α-L-Fucp-(1→6)-β-D-Glcp-(1→6)-β-D-Manp-(1→ and α-D-Xylp-(1→2)-α-L-Fucp-(1→5) -α-D-Araf-(1→6)-β-D-Galp-(1→ at O at H-4-arabinose and the terminal Galp residues, respectively. The MRC-5 cells induced by HO were used to explore the anti-ageing effect and its underlying mechanism of AAPS. It showed a potent anti-ageing activity, representing by the increased cell viability and β-Gal viability, prevented G1-phase cell-cycle arrest, and decreased mitochondrial membrane potential. The polysaccharides extracted from A. aegirita might be applied in functional food as anti-ageing ingredient.