The effects of tuna cooking juice hydrolysates by protease XXIII (PA) on cell growth and induction of apoptosis of MCF-7 cell line were determined. The PA hydrolysates showed antiproliferative activities up to 25% against MCF-7 cells, and the >2.5 kDa ultrafiltration fraction (PAH2.5) possessed the highest antiproliferative activity with an IC50 value of 1.39 mg/mL. PAH2.5 induced cell cycle arrest in S phase through the increases of p21 and p27, and decrease of cyclin A expression. Further, PAH2.5 also induced apoptosis of MCF-7 cells by downregulation of the expression of Bcl-2, PARP and caspase 9, and upregulation of the expression of p53, Bax and cleaved caspase 3. Two peptides were identified in PAH2.5 as KPEGMDPPLSEPEDRRDGAAGPK (2449.292 Da) and KLPPLLLAKLLMSGKLLAEPCTGR (2562.405 Da). Thus, tuna cooking juice may be a good protein source of antiproliferative peptides against MCF-7.