Extracellular polysaccharide from Pleurotus eryngii was sulfated; the optimum reaction conditions used pyridine as the reaction solvent, a temperature of 80°C, and a reaction time of 90 minutes. The structure and antioxidant activity of the sulfated and original polysaccharides (designated as SF-1 and Fr-1, respectively) were investigated. The main functional groups of polysaccharides were determined by Fourier transform infrared, and the chain conformation was determined by size-exclusion chromatography with laser light scattering and viscometry. Fourier transform infrared spectra indicated that the sulfated group was linked to the polymer. The molecular weight of SF-1 was much lighter than that of Fr-1, which suggested that degradation occurred during sulfation. The stable globular shape conformation of Fr-1 was changed to the random coil conformation of SF-1 in the aqueous solution. Moreover, the sulfated derivative SF-1 was found to have a better scavenging ability on hydroxyl radicals and DPPH free radicals than Fr-1. It seems that the chemical modification of P. eryngii polysaccharides by sulfation effectively enhances their antioxidant activity.