α-Hemolysin is a critical virulence factor in Staphylococcus aureus and its expression is largely controlled by the Agr-QS system. Nonetheless, the hemolysis phenotype and the regulation of the Agr-QS system in S. aureus still hold many mysteries. Our study finds that it is the expression of β- hemolysin rather than the agrA mutation that inhibits the function of the α-hemolysin in an important S. aureus strain RN4220 and a clinical strain presents a similar phenotype, which clarifies the misunderstood hemolytic phenotype and mechanism of S. aureus. Our findings highlight the interactions among different toxins and their biological roles, combined with QS system regulation, which is ultimately the true underlying cause of its virulence. This emphasizes the importance of considering the collaborative action of various factors in the infection process caused by this significant human pathogen.