Moringa oleifera Lamk. (M. oleifera) is an edible plant and used for traditional medicine formulation. Some bioactive phytochemicals found in M. oleifera leaves thus far were identified as quercetin, chlorogenic acid, astragalin, and kaempferol. The flavonoid kaempferol was reported to induce apoptosis in human HCT116 colon cancer cells. Here, we investigated the anti-proliferative activity present in the methanol extract from M. oleifera leaves toward human HCT116 colon cancer cells. Fractionation of the methanol extract from M. oleifera leaves by gel filtration chromatography on Sephadex LH-20 enabled us to find anti-proliferative and apoptosis-inducing activities. Treatment of HCT116 cells with each pooled fraction (pf1, pf2, or pf3) inhibited the cell proliferation in a dose-dependent manner, and the inhibitory activities contained in pf2 and pf3 were more potent than that in pf1. Compared with kaempferol, pf1, pf2, and pf3 were found to exhibit strong anti-proliferative effects on HCT116 cells. Futhermore, treatment with pf1 induced much larger numbers of cleaved caspase-3-positive cells than that with pf2 or pf3. The apoptosis-inducing activity found in each pooled fraction was higher than that of kaempferol. Cells treated with pf2 displayed the typical characteristics of apoptosis, such as membrane blebbing, nuclear condensation and apoptotic bodies, whereas cells treated with pf1 showed early apoptotic morphologies. In contrast, pf3 barely induced apoptosis despite its strong inhibition of cell proliferation. Taken together, these results suggest that, in addition to kaempferol, M. oleifera leaves may contain new substances having anti-proliferative and apotosis-inducing activities on HCT116 cells.