A carboxymethyl polysaccharide CMP33 (15.23 × 10⁴ Da) was isolated from edible and pharmaceutical mushroom Poria cocos using alkaline extraction followed by DEAE-52 and Saphadex-G200 + Saphadex-G150 column chromatographies. The structure analysis showed that CMP33 was composed of glucosyl residues containing a backbone chain of (1 → 3)-linked glucose residues and side chains of (1 → 6) and (1 → 2)-linked glucose residues, and possessed triple-helix structure. Bioassay results revealed that CMP33 displayed a dose-dependent inhibition on 5 cancer cells (HepG-2, MCF-7, SGC-7901, A549) in the range of 31.25-1000 μg/mL, but low cytotoxicity on normal liver cells L-O2. Moreover, CMP33 stimulated NO release and cytokine secretion (IL-1β, IL-6 and TNF-α), and also inhibited LPS-stimulated overproduction of NO, IL-6, TNF-α and IL-1β, in RAW264.7 cells. These results suggested that CMP33 possessed anticancer, anti-inflammation and immune-stimulation activities, and potential for developing as a bioactive ingredient in functional foods.