Abstract: The quenching effects of ethyl acetate extract of Lactobacillus pentosus DF9 as quorum sensing inhibitor on the quorum sensing (QS) of Vibrio parahaemolyticus were studied. Based on determining the effects of DF9-QSI on the growth of V. parahaemolyticus and V. harveyi BB170, the effects of DF9-QSI at sub-inhibitory concentration on QS signal molecule AI-2 activity, motility (swarming, swimming and twitching), biofilm formation and extracellular polysaccharide (EPS) synthesis of V. parahaemolyticus were investigated. The results showed that DF9-QSI had no effect on the growth of V. parahaemolyticus at the concentration lower than 0.8 mg/mL, but effectively reduced the AI-2 activity, and inhibited the movement, biofilm formation and EPS synthesis of V. parahaemolyticus in a concentration-dependent manner. The destructive effects of DF9-QSI on biofilms were further confirmed by the observations using optical microscope, scanning electron microscope and laser confocal microscope. These results indicated that DF9-QSI possessed AI-2 QS quenching activity, which might provide the theoretical basis for developing a novel QSI from lactic acid bacteria, and supply the potential microbial resources for controlling QS system of V. parahaemolyticus.