In the present study, bee pollen polysaccharides from Rosa rugosa (WRPP) were extracted and fractionated. WRPP were purified to neutral (WRPP-N) and acidic polysaccharides (WRPP-1, WRPP-2) with DEAE-Cellulose. WRPP-N were mainly composed of glucose, mannose, arabinose and galactose, indicating the existence of glucan, arabinogalactan (AG) and mannoglucan. WRPP-1 mainly consisted of rhamnose (3.0%), galacturonic acid (12.4%), galactose (24.7%) and arabinose (53.9%), and contained a large proportion of AGs. WRPP-2 consisted of rhamnose (7.8%), galacturonic acid (23.0%), galactose (15%) and arabinose (48.7%), while WRPP-2 contained more galacturonic acid compared to WRPP-1. WRPP-1 and WRPP-2 were composed by type I rhamnogalacturonan (RG-I), homogalacturonan (HG) and AG fragments, while WRPP-2 contained more HG and RG-I. All the fractions had significant anti-proliferative activity in HT-29 and HCT116 cells; the neutral and acidic fractions were shown to have significant synergistic effects which accounted for the antitumor activity of bee pollen polysaccharides from Rosa rugosa in vitro.