The present study aims to evaluate the anticancer activity of puerarin nanosuspensions in human colon cancer HT-29 cell line in vitro and in vivo. Puerarin nanosuspensions were prepared by the high-pressure homogenization (HPH) technique. The HT-29 cells were incubated with increasing concentrations of puerarin solution and nanosuspensions for indicated times. MTT evaluated cellular viability and investigated the effect of puerarin on cell proliferation of HT-29. Annexin V-FITC/PI staining method was conducted to determine the influences of the puerarin nanosuspensions on cell cycle and apoptosis. The in vivo anticancer activity of the puerarin nanosuspensions was observed in HT-29 cancer bearing mice. The puerarin nanosuspensions were well re-dispersed in aqueous media a mean diameter about 400-500 nm. Cytotoxicity assay, observation of morphological changes and early apoptosis revealed that the puerarin nanosuspensions could significantly enhance the in vitro anti-proliferation against HT-29 cells compared to the puerarin free solution. The prepared puerarin nanosuspensions in vivo evaluation showed higher anticancer efficacy and lower toxicity compared to the free solution, as shown by changes in tumor volumes, body weights, and survival rates. Based on these data, the potential of the puerarin nanosuspensions to serve as a cancer chemotherapeutic agent for colon cancer could be suggested.