Abstract: The strain (LP16) with high antibacterial activity was selected from the strong-flavor pit mud by double-layer AGAR diffusion method to explore its role in the pit mud. The results showed that strain LP16 was identified as Lactobacillus paracasei through morphological, physiological, biochemical characteristics and 16S rRNA. After the elimination of organic acid and hydrogen peroxide interference, the fermentation liquid still had obvious antibacterial effect, but the antibacterial activity lost. After being treated with trypsin, protease K, papain and pepsase, residual bacteriostat rates were 40.43%, 43.48%, 47.83% and 51.30%, respectively, indicating that the antibacterial substance contained bacteriocin with protein properties. The bacteriocin produced by the strain LP16 could maintain bacteriostatic activity after treated with high temperature, low pH (pH<5), ultraviolet irradiation and surfactant, and had a pH tolerance within the range of pH2~10. The antibacterial spectrum was wide, and it had obvious inhibitory effect on E. coli and Bacillus subtilis, as well as partial LAB, but it had no inhibitory effect on mold and yeast. Therefore, the bacteriocin produced by the bacteria could not only inhibit the growth of the pathogen, but also controled the excessive production of lactic acid.