A bacteriocin-like substance producing strain T12 was screened from 212 lactic acid bacteria isolated from intestine of healthy pigs by paper disk test and was identified as Lactobacillus. The supernatant of T12 exhibited strong inhibitory activity against the gram-positive bacteria, including Micrococcus lysodeikticus, Staphylococcus aureus, Bacillus subtilis and Bacillus cereus. A strong activity was also observed against the gram–negative bacteria and some eumycete, such as Escherichia coli, Salmonella typhimurium and Fusarium graminearum. Through detection of its appearance, physiological and biochemical characteristics and 16S rDNA gene sequence homology analysis, it was identified as Lactobacillus animalis. Eliminated the organic acids and hydrogen peroxide the fermented production exhibited strong inhibitory activity against indicat bacterias still; the inhibitory activity was totally lost after treatment with protease; The bacteriocin-like substance was heat-stable even at the autoclaving temperature (121°C for 20 min) and was active over a wide pH range (3.5~5.5). The protein nature and restricted spectrum of the substance points to its being bacteriocin-like substance.