The crude polysaccharides (UA) were obtained from Urtica angustifolia by hot water extraction, 80% ethanol precipitation and deproteination by using sevage reagent in a yield of 1.6%. The polysaccharides were fractionated into six fractions. Their sugar compositions and structures were analyzed by high performance liquid chromatography (HPLC) and nuclear magnetic resonance spectrometer (NMR). The HPLC analysis results showed that UA contain two neutral polysaccharide fractions and four acidic fractions. The neutral fractions are mainly composed of glucose (Glc), and the acidic fractions are mainly composed of galacturonic acid (GalA). The NMR results showed that the neutral fractions are starch-like polysaccharides and the acidic fractions are rhamnogalacturonan (RG)-I domian containing pectin. The anti-fatigue activity of the polysaccharides was evaluated in mice and the results showed U. angustifolia polysaccharides increased the swimming time, and the average swimming time of the low doses (LD), middle doses (MD) and high doses (HD) group were increased by 85.4, 114.6 and 151.2%, respectively. The blood urea nitrogen content of the LD, MD and HD group was reduced by12.8, 13.6 and 19.8% compared with that of control group, respectively. The hepatic glycogen of every treated group was higher than that of control group, increasing rates were 87.0, 469.6, and 747.8%, respectively.