A novel polysaccharide (AGP-32) from the gonad of Haliotis discus hannai Ino was isolated using a protease-assisted process and successive ion-exchange and gel-filtration chromatography. The backbone of AGP-32 was determined using hydrolysis with trifluoroacetic acid. FTIR, NMR, and methylation analysis, and periodate oxidation and Smith degradation analysis revealed that the AGP-32 backbone mainly consisted of (1→6)-linked mannose, (1→3)-linked galactose, and (1→3)-linked glucose in a proportion of 2:3:1. An in vitro cell assay indicated that AGP-32 promoted mice splenic lymphocyte proliferation by 26% at a concentration of 50 μg/mL. AGP-32 had an effect on immune protection and is a candidate for consideration as a functional food.