Mushrooms have been used since centuries for the treatment of different diseases and efforts were made to isolate different compounds from the mushrooms responsible for medicinal activities. Among the mushroom genus Pleurotus is worldwide in distribution due to their medicinal properties such interventions ameliorate hepatic lipid profiles, reduce inflammation and oxidative stress, noticeable antioxidant, antiinflammatory, antimicrobial, antitumor and analgesic properties. These properties are attributed mainly to polysaccharides synthesized by Pleurotus. The aim of current study is to improve of Exopolysaccharide production by Pleurotus sapidus through manipulation of different culture and environmental conditions using response surface methodology (RSM). Plackett–Burman design indicated that; shaking, starch, and incubation temperature are the most effective parameters. Box–Behnken RSM indicated that; the optimum concentration for each parameter was: 148.9 rpm for shaking, 30.1g/l starch, and 25.2 ºC for incubation temperature. After production, EPS was extracted, purified, identified using Fourier transform infrared (FTIR), Nuclear Magnetic Resonance (¹HNMR) and High Performance Liquid Chromatography (HPLC). The result indicated that; it consisting of β-(1→3) glucans, β-(1→6) glucans branched, Chitin, Mannans, and Galactans with different concentrations in EPS. Exopolysaccharides extracted from Pleurotus sapidus showed antitumor, antiviral, antibacterial, anticoagulant and antioxidant activity.