Anti-proliferative and pro-apoptotic activities of fractions of Pleurotus ostreatus were examined using HT-29 colon cancer cells in vitro. A hot-water-soluble fraction of the mycelium of the liquid cultured mushroom was partially isolated and chemically characterized as a low-molecular-weight alpha-glucan. HT-29 cells were exposed to the different isolates and significant inhibition of proliferation was obtained in a dose-dependent manner. Proliferation inhibition was shown to be the result of apoptotic induction because the pro-apoptotic molecules Bax and cytosolic cytochrome-c were upregulated. Fluorescence-activated cell sorter analyses of polysaccharide-treated HT-29 cells showed a high percentage of Annexin-positive cells. Here, we describe a newly identified low-molecular-weight alpha-glucan with promising anti-tumorigenic properties, and demonstrate its direct effect on colon cancer cell proliferation via induction of programmed cell death.