The objectives of this study were to investigate the effect of fermentation medium on the hydroxyl radical scavenging activity of exopolysaccharides from Inonotus obliquus by response surface methodology (RSM). A two-level fractional factorial design was used to evaluate the effect of different components of the medium. Corn flour, peptone, and KH<sub>2</sub>PO<sub>4</sub> were important factors significantly affecting hydroxyl radical scavenging activity. These selected variables were subsequently optimized using path of steepest ascent (descent), a central composite design, and response surface analysis. The optimal medium composition was (% w/v): corn flour 5.30, peptone 0.32, KH<sub>2</sub>PO<sub>4</sub> 0.26, MgSO<sub>4</sub> 0.02, and CaCl<sub>2</sub> 0.01. Under the optimal condition, the hydroxyl radical scavenging rate (49.4%) was much higher than that using either basal fermentation medium (10.2%) and single variable optimization of fermentation medium (35.5%). The main monosaccharides components of the RSM optimized polysaccharides are rhamnose, arabinose, xylose, mannose, glucose, and galactose with molar proportion at 1.45%, 3.63%, 2.17%, 15.94%, 50.00%, and 26.81%. © The Korean Society for Microbiology and Biotechnology.