In the present study, ultrasound-assisted extraction (UAE) of crude polysaccharides (PSP) from pumpkin seeds was optimized by response surface method (RSM). The polysaccharide yield (2.29 ± 0.14%), which agreed closely with the theoretical predicted value 2.40%, was obtained under the optimal extraction conditions: extraction time 24 min, extraction temperature 50 °C, ultrasonic power 347 W, and liquid to solid ratio 23 mL/g. After further purification by two-step column chromatography, a novel polysaccharide (PSP-1) was isolated from pumpkin seeds. PSP-1 was composed of mannose, glucose, and galactose in a molar ratio of 1.00:4.26:5.78 with molecular weight of 3728 g/mol. 1D and 2D NMR spectroscopy analysis revealed that the backbone of PSP-1 was mainly formed by β→6)-β-d-Galp-(1→, →6)-α-d-Glcp-(1→, and →3,6)-β-d-Manp-(1→ with branching at O-3 and O-6 of →3,6)-β-d-Manp-(1→. Branch linkages were composed of α-d-Glcp-(1→ and →4)-α-d-Galp-(1→.