ASP is a serine protease secreted by Aeromonas sobria. ASP cleaves various plasma proteins, which is associated with onset of sepsis complications, such as shock and blood coagulation disorder. To investigate a host defense mechanism against this virulence factor, we examined the plasma for ASP inhibitor(s). Human plasma inhibited ASP activity for azocasein, which was almost completely abolished by treating plasma with methylamine, which inactivates α2-macroglobulin (α2-MG). The ASP-inhibitor complex in ASP-added plasma was not detected by immunoblotting using anti-ASP antibody; however, using gel filtration of the plasma ASP activity for an oligopeptide, the ASP substrate was eluted in the void fraction (Mw>200 000), suggesting ASP trapping by α2-MG. Indeed, human α2-MG inhibited ASP azocaseinolytic activity in a dose-dependent manner, rapidly forming a complex with the ASP. Fibrinogen degradation by ASP was completely inhibited in the presence of α2-MG. α1-Protease inhibitor, antithrombin, and α2-plasmin inhibitor neither inhibited ASP activity nor formed a complex with ASP. Surprisingly, ASP degraded these plasma serine protease inhibitors. Thus, α2-MG is the major ASP inhibitor in the human plasma and can limit ASP virulence activities in A. sobria infection sites. However, as shown by fluorescence correlation spectroscopy, slow ASP inhibition by α2-MG in plasma may indicate insufficient ASP control in vivo.