The study was aimed to evaluate the antimicrobial activity of glycyrrhiza flavonoid versus S. aureus, and further investigate the impact of subinhibitory concentrations of glycyrrhiza flavonoid on the production of α-hemolysin in S. aureus. A broth microdilution method was employed to determine the minimal inhibitory concentrations (MIC) of glycyrrhiza flavonoid against 6 S. aureus strains. Hemolysis assay, Western-blot and Real-time RT-PCR assays were performed to the study the influence of subinhibitory concentrations of glycyrrhiza flavonoid on the production of α-hemolysin at phenotypic, expressional and transcriptional levels, respectively. The data shown that glycyrrhiza flavonoid has great anti- S. aureus activity with MICs ranged from 4 to 16 μg/mL; and subinhibitory concentrations of glycyrrhiza flavonoid inhibit the expression of α-hemolysin in a concentration-dependent fashion.