A novel bacteriocin, plantaricin ZJ008 produced by Lactobacillus plantarum ZJ008 isolated from fresh milk, was purified by XAD 2, cation exchange chromatograph, gel chromatograph, and RP-HPLC. Mass spectrometry based on MALDI-TOF indicated that the bacteriocin had a molecular of 1334.77 Da. Only five of twenty amino acids could be identified by Edman degradation. This bacteriocin was highly thermostable (121 °C, 30 min) and exhibited narrow pH stability (pH 4.0–5.0). It was sensitive to α-Chymotrypsin, trypsin, papain, and pepsin. However it still had 80% of activity after treatment by proteinase K. The action mode of this peptide functioned as bactericidal, but it did not lead to lysis of cells. This bacteriocin exhibited broad-spectrum antimicrobial activity against tested Gram-positive and Gram-negative bacteria, especially Staphylococcus spp. These results suggested that this bacteriocin appears potentially very useful to control and inhibit Staphylococcus spp. in the food industry.