6-Acetonyldihydrochelerythrine (1), a benzophenanthridine alkaloid, isolated from the methanol extract of Zanthoxylum capense, displayed potent cytotoxic activity in human HCT116 and SW620 colon carcinoma cells, to a higher extent than 5-fluorouracil (5-FU), the cornerstone chemotherapeutic agent in colon cancer. Cytotoxicity of 1 was evaluated by MTS, lactate dehydrogenase (LDH), and Guava ViaCount assays. Interestingly, 1 significantly induced cytotoxicity in both cell lines, leading to a significant increase in LDH release, as compared to 5-FU. Further, Guava ViaCount flow cytometry assays demonstrated that 1 significantly increased cell death, as shown by the presence of a significantly higher population of apoptotic cells in both cell lines, as compared to cells exposed to 5-FU. Furthermore, evaluation of nuclear morphology by Hoechst staining of 1-treated HCT116 and SW620 cells confirmed flow cytometry results, demonstrating a marked induction of apoptotic cell death by 1, again to a further extent than that elicited by 5-FU. In addition, immunoblot analysis to ascertain the molecular events triggered by 1 exposure was performed. The results show that 1 exposure reduced the steady-state expression and activation of the pro-survival proteins ERK5 and Akt and increased the steady-state expression of p53 in both HCT116 and SW620 cells. Changes in ERK5 or Akt activation can be ascertained by evaluating the ratio of p-ERK5/ERK5 or p-Akt/Akt. In addition, exposure to 1 reduced expression of XIAP, Bcl-XL, and Bcl-2, while increasing the cleavage of poly(ADP-ribose) polymerase in both cell lines. Collectively, the data indicate that 6-acetonyldihydrochelerythrine (1) is a potent inducer of apoptosis in HCT116 and SW620 cell lines, highlighting its potential relevance in colon cancer.