Enzyme assisted extraction was used to extract the polysaccharides from pumpkin seeds (PSP) and the extraction parameters were optimized by response surface methodology (RSM). Under the optimum experimental parameters: Extraction temperature of 60 °C, extraction time of 43 min, enzyme concentration of 2.5%, and pH of 6.0, the yield of PSP was 3.22 ± 0.04%, which was in close agreement with the predicted value (3.24%). After further purification on anion exchange column and gelfiltration column, a novel purified polysaccharide (PSPE) with molecular weight of 16,700 g/mol was obtained. PSPE was mainly composed of mannose, galactose and glucose in the molar ratio of 1.00:3.84:1.62. NMR spectra analysis showed that the major backbone of PSPE consisted of →4)-α-d-Glcp-(1→, →4)-β-d-Manp-(1→, →3,6)-β-d-Glap-(1→, and β-d-galactose.