Maca (Lepidium meyenii Walp) polysaccharides (MP) with purity of 99.2% were obtained to investigate their structural characteristics and antifatigue effect in vivo. The physicochemical properties of MP were analyzed through high-performance gel filtration chromatography, IR, monosaccharide composition, methylation, GC-MS, and NMR analyses. The antifatigue effect of MP was evaluated by using a mouse weight-loaded swimming model. MP is an acidic heteropolysaccharide with an average molecular weight (M ) of 793.5 kDa. It is composed of D-GalA: D-Glc: L-Ara: D-Man: D-Gal: L-Rha = 35.07:29.98:16.98:13.01:4.21:0.75 (mol, %). The findings revealed that MP contained β-1,3-Galp(A), β-1,3-Glcp, and α-1, 3-Manp linked alternatingly to form a backbone (5:4:1). MP (above mid-dosage 50 mg/kg bw/d) could effectively elongate swimming durations and accelerate average swimming speeds (within the 1st 5 min) of mice (P < 0.05) and improve the serous biochemical parameters of mice. Compared with the control model, high-dosage (100 mg/kg bw/d) MP treatment could significantly enhance glutathione peroxidase and creatine kinase activities (P < 0.05) and decreased lactate dehydrogenase activity (P < 0.01). High-dosage MP could significantly reduce the levels of blood urea nitrogen, lactic acid, and malondialdehyde (P < 0.05). MP is an acidic polysaccharide with a high D-GalA content, which could be responsible for the antifatigue effect of maca.