The peptides produced enzymatically from various plants have shown various biological activities including cytotoxicity. Different types of cytotoxic peptides have been reported from the seeds and leaves of Violaceae, Rubiaceae and Annonaceae families. In this study, we report purification and characterization of peptide(s) showing cytotoxic activity against A549 and HeLa cancer cell lines from the seeds of Polyalthia longifolia (Annonaceae). Seed proteins of P. longifolia were extracted and hydrolyzed using trypsin. The enzyme hydrolysate was applied on to a Sephadex G10 column and eluted using Tris-HC1 buffer (pH 7.5). Two fractions F1 and F2 were obtained, of which F2 showed significant cytotoxic activity against lung (A549) cancer cells at 10 microg/mL and cervical (HeLa) cancer cell lines at 30 microg/mL, as revealed by the MTT assay. DNA fragmentation was observed in the tested cancer cell lines treated with F2 peptide at a concentration of 10microg/mL and 30 pg/mL, respectively. Further, increased number of apoptotic cells was observed in sub-G0 phase of cell cycle of A549 and HeLa cell lines, when treated with 10 microg/mL and 30 microg/mL of F2, as revealed by the flow cytometric analyses. FTIR spectrum of F2 peptide detected the presence of stretching vibrations of carboxylic acid OH residue with peak at 3420 cm-and carbonyl (C=O) groups at 1636 cm-1, respectively. RP-HPLC analysis of F2 peptide showed a single peak at a retention time of 12.8 min detected at 280 nm, depicting the purity of F2 to be more than 90%. LC-ESI-MS/MS analysis showed the average theoretical mass of F2 to be 679.8 using m/z ratios. In conclusion, the findings suggest that F2 peptide is an effective inducer of apoptosis of cancer cells, thus offers an important strategy in the development of cancer therapeutics.