A novel acidic polysaccharide (RTFP-3) was isolated and purified from Rosa roxburghii Tratt fruits. Structural analyses revealed that RTFP-3 is a homogeneous polysaccharide with a molecular weight of 67.2 kDa and mainly consists of arabinose (37.20%), galactose (34.40%), glucose (10.02%) and fucose (18.30%). The structural chain of RTFP-3 was proven to be composed of →6)-α-d-Galp-(1→, →4)-α-d-GalpA-(1→, →5)-α-l-Araf-(1→, →4)-β-d-Glcp-(1→, β-d-Glcp-(1→ and →3,4)-β-l-Fucp-(1→ residues by periodate oxidation-Smith degradation, methylation, and 1D- and 2D-NMR analyses. Bioassay results showed that RTFP-3 exerted favorable mixed type inhibitory activities against α-glucosidase, and its half inhibitory concentration (IC50) value was determined as 4.15 mg mL-1. RTFP-3 showed static type quenching of the intrinsic fluorescence of α-glucosidase, during which the binding process induced the conformational change of α-glucosidase. These results suggest that RTFP-3 has the potential to be a novel α-glucosidase inhibitory agent for use in functional foods.