Bovine subclinical mastitis is an inflammation of the mammary gland caused by bacterial intramammary infection, accounting for large economic losses. Treatment of subclinical mastitis is not suggested for lactating cows due to the risk of milk contamination. The objectives of this study were to evaluate an antimicrobial peptide, nisin, in the treatment of subclinical mastitis in lactating cows. A total of 90 lactating Holstein cows with subclinical mastitis were randomly divided into nisin-treated (n = 46) and control (n = 44) groups. In the nisin-treated group, cows received an intramammary infusion of nisin at a dose of 2,500,000 IU once daily for 3 days while the control cows received no treatment. Milk samples were collected from the affected mammary quarters before treatment and 1 and 2 weeks after treatment for analyses of bacteria, somatic cells, and N-acetyl-beta-D-glucosaminidase (NAGase). Results indicated that nisin therapy had bacteriological cure rates of 90.1% for Streptococcus agalactiae (10 of 11), 50% for Staphylococcus aureus (7 of 14), 58.8% for coagulase-negative staphylococci (7 of 17), and 65.2% for all cases (30 of 46). Meanwhile, only 15.9% (7 of 44) of untreated cows spontaneously recovered. NAGase activity in milk samples and the number of mammary quarters with a milk somatic cell count of > or =500,000/ml were significantly decreased after nisin treatment while no significant changes took place in the control group. Because of its therapeutic effects on bovine subclinical mastitis, as well as its safety in humans, nisin deserves further study to clarify its effects on mastitis caused by different pathogens.