Colon cancer is one of the common types of digestive malignancy. The efficacy of the first-line chemotherapy drug for colon cancer, fluorouracil (5-FU), remains limited in clinical settings due to poor efficacy and significant side effects. In the present study, the anticancer activity of an active compound from Pulsatilla chinensis extracts, Pulsatilla saponin A (PsA), was isolated and examined in vitro and in vivo. It was demonstrated that PsA significantly inhibited the growth of human colon cancer HT-29 cells. This inhibitory activity was also observed when the compound was tested in a colon cancer xenograft mouse model. Additionally, the synergic antitumor effects of PsA and 5-FU on colon cancer cells were observed. Using annexin V and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling assays, it was demonstrated that levels of apoptosis induction in HT-29 cells treated with PsA or 5-EU were significantly increased compared with the untreated control cells (P<0.05). Western blot analyses were then performed, and the results revealed an increase in tumor protein 53 and cleaved caspase 9, and a decrease in B-cell lymphoma 2 protein expressions in PsA and PsA + 5-FU treated colon cancer cells compared with the vehicle-treated (PBS) cells. In summary, PsA exhibited anticancer activity in human colon cancer cells in vitro and in vivo, in isolation and synergistically with 5-EU, through apoptosis induction.