Treatment of cells with the anti-cancer drug camptothecin (CPT) induces topoisomerase I (Top1)-mediated DNA damage, which in turn affects cell proliferation and survival. In this report, we demonstrate that treatment of the wild-type HCT116 (wt HCT116) human colon cancer cell line and the isogenic p53-/- HCT116 and p21-/- HCT116 cell lines with a high concentration (250 nM) nm) of CPT resulted in apoptosis, indicating that apoptosis occurred by a p53- and p21-independent mechanism. In contrast, treatment with a low concentration (20 nM) of CPT induced cell cycle arrest and senescence of the wt HCT116 cells, but apoptosis of the p53-/- HCT116 and p21-/- HCT116 cells. Further investigations indicated that p53-dependent expression of p21 blocked apoptosis of wt HCT116 cells treated with 20 nM, but not 250 nM CPT. Interestingly, blocking of the apoptotic pathway, by Z-VAD-FMK, in p21-/- HCT116 cells following treatment with 20 nM CPT did not permit the cells to develop properties of senescence. These observations demonstrated that p21 was required for senescence development of HCT116 cells following treatment with low concentrations of CPT.