Actinobacillus pleuropneumoniae is the pathogen of porcine contagious pleuropneumonia causing great economic losses globally. LuxS is known to be involved in the interspecies quorum sensing by mediating the production of AI-2 signal molecule and play important roles in regulating various behaviors of bacteria. In this study, A. pleuropneumoniae was tested to be able to produce AI-2 like molecule. A luxS deletion mutant strain was constructed by homologous replacement, and its growth features, biofilm formation and virulence were characterized. The luxS mutant showed obvious growth deficiency when cultured in the serum-free medium. Biofilm formation of the mutant strain was significantly enhanced. In a mouse infection model, the 50% lethal dose of the mutant strain was increased up to 96-folds, and the ability to colonize in different mouse tissues was significantly decreased. Down-regulation of apxIIA, a major virulence factor of A. pleuropneumoniae, may contribute to the virulence attenuation of the mutant strain. Our data demonstrated that LuxS is functional in the AI-2-mediated quorum sensing in A. pleuropneumoniae and LuxS may regulate different behaviors including biofilm formation and virulence of this important pathogen.