In our previous study, Abrus agglutinin showed antitumor activity both native and heat-denatured condition in mouse model. The purpose of this study is to explore the presence of anticancer peptide in agglutinin, and to elucidate the mechanism of its activity in vitro. A tryptic digested Abrus agglutinin peptide fractions obtained from 10-kDa molecular weight cut off membrane permeate (10kMPP), was found to have selective antiproliferative activity (1–10μg/ml) on several tumor cell lines in vitro without having any cytotoxic effect on normal cell lines with dose of 100μg/ml. Analysis of the growth inhibitory mechanism in HeLa cells revealed nuclear fragmentation and condensation with appearance of the sub-G 0 /G 1 peak indicative of apoptosis. Furthermore, the peptide fraction induced the apoptosis signal via generation of reactive oxygen species and decrease in the Bcl-2/Bax ratio thereby inducing mitochondrial permeability transition with consequent activation of caspase-3, finally leads to DNA fragmentation, and the hallmark of apoptosis. LC-MS/MS analysis reflected the molecular masses of peptides in 10kMPP were in the range of 500Da to 3000Da. The significant antitumor activity of 10kMPP deserves further laboratory and in vivo experimentation.