This study was conducted to enrich and purify the active constituent in Polygonum cuspidatum, and to study its anticancer activity. Extraction conditions of Polygonum cuspidatum were optimized by investigating the macroporous resin model, flow rate and ethanol concentration. MTT assay and electron microscopy were used to analyze the inhibitory effect of Polygonum cuspidatum extract on human SW480 cells. During the investigation of enrichment and purification process, the optimal conditions for enrichment of Polygonum cuspidatum extract with D101 macroporous resin were identified to be a 3-fold amount of 75% ethanol, and a flow rate of 2 BV/h. Under electron microscope, nuclear morphological changes are seen in SW480 cells treated with Polygonum cuspidatum extract; apoptotic characteristics are significant, and apoptotic bodies are formed eventually. MTT assay results showed that the inhibitory effect of Polygonum cuspidatum extract on SW480 cells increases with the increase of concentration and prolongation of action time within the experimental concentration range, which proves a dose- and time-dependence of action. Polygonum cuspidatum extract has a marked inhibitory effect on human colon cancer SW480 cells.