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In this study, crude peptide fractions from Hanwoo loins were released by injecting with proteolytic enzymes [no enzymes (control); protease type XIII (E1); thermolysin (E2); and combination of E1 and E2 (E3)] and their bioactivities were determined. The peptides derived from E2-injected Hanwoo loin exhibited the highest angiotensin I–converting enzyme (ACE) inhibitory activity and vitamin C equivalents antioxidant capacity among the treatments. The released peptide by treatment of E2 and E3 had similar (P > 0.05) inhibitory activity in HT29 cancer cell viability compared with luteolin as a positive control and non-cytotoxic effect on normal cell (3T3-L1). Therefore, the released peptide fraction from thermolysin (E2)-injected Hanwoo beef might contain potent bioactive peptides with ACE inhibitory and antioxidative activity and inhibition effect on certain cancer cell viability.