In this study, the antitumor properties and the possible molecular mechanisms of Cyclocarya paliurus polysaccharide (CP) and its phosphorylated derivative (P-CP) on CT-26 mouse colon carcinoma cells were investigated. Results found that CP had high inhibition ratio against CT-26 cells. The flow cytometry results found that CP treatment could cause the intracellular acidification, arrest the cell cycle in the S phase and increase reactive oxygen species generation. Additionally, CP treatment triggered mitochondrial membrane potential depolarization and Ca2+ overloading, and broke down the balance of antioxidant system, Na+/K+-ATPase and Ca2+-ATPase. Further analysis found CP induced cell apoptosis through improving the activities of caspase-3 and caspase-9, and increasing the level of cytochrome C. Furthermore, the comparative study of antitumor effect on CT-26 cells displayed that the phosphorylation enhanced antitumor activities of polysaccharides. These results suggest CP is a potential natural therapeutic agent for colon cancer and phosphorylation represents an effective method of enhancing the antitumor activity of CP.