In this investigation we developed an optimal fermentation medium to produce an exopolysaccharide (EPS) with antioxidant activity from Rigidoporus microporus. Lactose and tryptone were chosen as the optimal carbon and nitrogen sources, respectively, for EPS production, with a 6-day cultivation cycle. After removing proteins through the use of the Sevag method, one EPS fraction was purified from the culture filtrates by gel filtration chromatography on a Sepharose CL-6B column. The preliminary chemical structure of the EPS fraction was determined by Fourier transform infrared spectroscopy, gas chromatography-mass spectrometry, and nuclear magnetic resonance. The results indicated that the main structure of the EPS consisted of β-glucopyranose and mannopyranose. Furthermore, conformational parameters such as molecular weight (Mw), intrinsic viscosity ([η]), mean-square radii of gyration (Rg), and hydrodynamic radius (Rh) of the EPS were characterized using a size exclusion chromatography-multiangle light scattering-refractive index viscometry method. It showed that the EPS was a kind of water-soluble polysaccharide with a moderate molecular weight (34.1 × 104) and a flexible, linear random coil chain structure. The antioxidant activity tests suggested that the EPS has great potential application as a natural antioxidant material in foods and medicines.