Bacterial populations use a cell-to-cell communication system to coordinate community-wide regulation processes, which is termed quorum sensing (QS). Autoinducer-2 (AI-2) is a universal signal molecule that mediates inter- and intraspecies QS systems among different bacteria. In this study, the effects of exogenous addition of AI-2 synthesized in vitro on physiological behaviors and proteome were investigated in lactic acid bacteria strains. Exogenous AI-2 had a concentration-dependent effect on the 8-3 cell density. There was no significant influence on biofilm formation and individual morphology of cells upon 60 μM AI-2 addition in 8-3 and 2-1. However, it improved the acid and alkali resistance of 8-3. With the addition of AI-2, 15 differentially expressed proteins were identified in 8-3, which participate in RNA transport signaling, RNA polymerase, ribosome, oxidative phosphorylation, cysteine and methionine metabolism, pyrimidine metabolism, ATP-binding cassette (ABC) transporters, purine metabolism, biosynthesis of the amino acid pathway, etc. Among them, the expression of 5-methylthioadenosine/-adenosylhomocysteine nucleosidase, which is known to be involved in AI-2 synthesis and cysteine and amino acid metabolism, was upregulated. These findings will lay the foundation to clarify the mechanism of cell-to-cell communication and bacterial physiological behaviors mediated by AI-2.