Streptococcus pyogenes is a major human pathogen causing more than 700 million infections annually. As a successful pathogen, S. pyogenes produces many virulence factors that facilitate colonization, proliferation, dissemination, and tissue damage. Serum opacity factor (SOF), an extracellular protein, is one of the virulence factors made by S. pyogenes. The underlying mechanism of how SOF contributes to virulence is not fully understood. SOF has two major features: (i) it opacifies host serum by interacting with high-density lipoprotein, and (ii) it inhibits beta-hemolysis on blood agar. In this study, we demonstrate that the domain of SOF essential for opacifying serum is also essential for SOF-mediated beta-hemolysis inhibition and SOF-mediated virulence. Our results shed new light on the molecular mechanisms of SOF-host interaction.