To explore the antibacterial mechanism of nisin Q13 against Lactobacillus bulgaricus LMG-1, the purity and molecular mass of nisin Q13, produced by Lactococcus lactis Q13 and purified by ammonium sulfate precipitation, ultrafiltration and dextran gel column chromatography, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the effects of nisin Q13 on the cell membrane permeability, intracellular enzyme activity and cell morphology of L. bulgaricus were evaluated. The results showed that the optimal saturation level of ammonium sulfate for nisin Q13 precipitation was 60%. After ultrafiltration, only one fraction, whose molecular mass was greater than 10 kDa, had an antibacterial effect. The fraction was further purified by dextran gel column chromatography, yielding two peaks with antibacterial activity. The polyacrylamide gel electrophoretogram showed only one protein band with a molecular mass of about 17.5 kDa, which may be a polymer of nisin. The antibacterial mechanism of nisin Q13 may be related to the fact that it can cause the formation of holes in the cell membrane of the target cells, increase the permeability of the cell membrane and destroy the integrity of the cell membrane, leading to the leakage of cellular contents, reducing the contents of key enzymes such as Na⁺/K⁺-ATPase and alkaline phosphatase (AKP), affecting the normal energy metabolism of cells, and eventually resulting in cell death.