The purpose of this work was to fractionate the complex exopolysaccharide (EPS) from a medicinal fungus Ophiocordyceps sinensis Cs-HK1 based on the molecular weight (MW) range and to assess the in vitro anti-inflammatory activity of different EPS fractions in THP-1 cell culture. The lower MW fraction (EPS-LM-1) showed a much higher anti-inflammatory activity. EPS-LM-1 was identified as a heteropolysaccharide consisting of mannose, glucose, and galactose residues with an average MW of 360 kDa. EPS-LM-1 significantly inhibited the lipopolysaccharide-induced inflammatory responses with the effective concentrations for 50% inhibition below 5 µg/mL on a few major proinflammatory markers. With such a notable in vitro anti-inflammatory activity, EPS-LM-1 is a promising candidate for the development of a new anti-inflammation therapy.