Bacteriocin KU24 produced by Lactococcus lactis KU24 exhibited an inhibitory effect against methicillin-resistant Staphylococcus aureus (MRSA). Bacteriocin KU24 was inactivated by protease XIV, showing that it has a proteinaceous nature on S. aureus ATCC 33591. Also, bacteriocin KU24 exhibited a strong heat stability (121 °C for 15 min) and pH stability (pH 3 to 9). The mode of inhibition was determined for S. aureus ATCC 33591 by treatment of 0, 250, and 500 AU/mL of bacteriocin KU24. S. aureus ATCC 33591 was inhibited by added bacteriocin KU24, while control was increased. The cell membranes of S. aureus ATCC 33591 were damaged with treatment of 500 AU/mL of bacteriocin KU24. Also, bacteriocin KU24 inhibited the occurrence of mecA gene, the methicillin resistance gene in S. aureus ATCC 33591. Bacteriocin KU24 was purified by C18 Sep-Pack column, cation exchange chromatography, and gel filtration chromatography, and molecular mass is approximately 6.5 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These results demonstrate that bacteriocin KU24 can be used as an alternative antimicrobial agent for the treatment of infection of MRSA in the food industry.