Autoinducer-2 (AI-2) is a universal signal molecule mediating intra- and interspecies communication among bacteria. AI-2 is a byproduct of the LuxS enzyme during the catabolism of S-adenosylhomocysteine and plays critical roles in regulating various behaviors of bacteria. In our previous study, the function of LuxS in AI-2 production was verified in Streptococcus suis (SS). Decreased levels of SS biofilm formation and host-cell adherence as well as the inability to produce AI-2 were observed in SS having a luxS mutant gene. In this study, exogenous addition of a low concentration of AI-2 synthesized in vitro was found to promote biofilm formation and host-cell adherence. However, higher concentrations of AI-2 inhibited SS biofilm formation and host-cell adherence. Real-time PCR results showed that the mRNA level of virulence factors of SS biofilm, gdh, cps2, sly, and mrp increased and ef, fbps, and gapdh decreased with increasing AI-2 concentrations. These findings demonstrated that AI-2 supplemented exogenously acted as a concentration-dependent signaling molecule to regulate SS biofilm formation, host-cell adherence, and transcription levels of many virulence genes.