In this study, Porphyra haitanesis protein was extracted and hydrolysed with trypsin. The gel filtration chromatography was employed to separate peptides from protein hydrolysate. Three polypeptides were isolated, which exhibited anti-proliferation activities on five cancer cells (MCF-7, HepG-2, SGC-7901, A549 and HT-29), the 1050 values were comprised within 191.61 and 316.95 mu g/mL. Subsequently, two new peptides VPGTPKNLDSPR and MPAPSCALPRSVVPPR were identified, the former displayed good inhibition activities on cancer cells MCF-7 and HepG-2, with the IC50 values of 200.97 mu g/mL and 276.85 mu g/mL, respectively, but low inhibition on normal liver cells LO-2 (7.1%), at 500 mu g/mL. Moreover, flow cytometry confirmed that cell cycle was arrested in G0/G1 phase and apoptosis was induced in MCF-7 cells treated with the synthesized peptide VPGTPKNLDSPR. Taken together, Porphyra haitanesis-derived polypeptides possessed anti-proliferation activities by arresting cell cycle and inducing apoptosis in cancer cells, which could be potential in food and pharmaceutical applications.