Background: Recently, much attention has been paid to natural products owing to their effective anticancer effects with relatively low toxicity, especially Inonotus obliquus (I. obliquus).Results: Polysaccharides with different molecular weights were filtered from water extract of I. obliquus using grading membrane filtration method. IOP60b (10 kDa ≤ molecular weight ≤ 30 kDa) was found to have the highest yield and the highest inhibition ratio of HT-29 cancer cells, therefore it was chosen to evaluate the apoptotic effect of HT-29 cancer cells. After treated with different concentrations of IOP60b, morphological changes including cell shrinkage and nuclear condensation and DNA fragmentation in HT-29 cancer cells were observed, and cells in early apoptosis and late apoptosis significantly (p<0.05) increased in a dose-dependent manner by arresting cell cycle at G0/G1 phase. To further explore the underlying mechanism, RT-PCR and Western blotting were applied. Results indicated that both Bcl-2 family and Caspase family were involved in the process of apoptosis regulation and IOP60b induced cellular apoptosis via upregulation of Bax/Bcl-2 ratio and activation of Caspase-3.Conclusion: These data suggested that IOP60b might be the potential candidate for the clinical prevention and treatment of colorectal cancer.