The anticancer effect and apoptotic mechanism of a novel indole derivative AC-264, a lead derived from a chemical library, were investigated in human promyelocytic leukemia HL-60 cells. HL-60 cells treated with AC-264 at various concentrations showed the morphological features of apoptosis, such as plasma membrane blebbing and cell shrinkage. AC-264 exhibited cytotoxic effect in various cancer cell lines with different degrees of potency. Especially, AC-264 was effective on increasing the population of apoptotic cells in HL-60 cells, as detected by the number of cells stained with Annexin V and PI. Furthermore, AC-264 activated caspase-3 enzyme activity and induced internucleosomal DNA fragmentation. These results indicated that AC-264 produces anti-cancer effect via apoptotic cell death by activating caspase-3 and inducing internucleosomal DNA fragmentation in HL-60 cells.