Many pathogenic enveloped viruses, including HIV-1, escape complement-mediated virolysis by incorporating host cell regulators of complement activation into their own viral envelope. The presence of complement regulators including CD59 on the external surface of the viral envelope confers resistance to complement-mediated virolysis, which may explain why human pathogenic viruses such as HIV-1 are not neutralized by complement in human fluids, even in the presence of high Ab titers against the viral surface proteins. In this study, we report the development of a recombinant form of the fourth domain of the bacterial toxin intermedilysin (the recombinant domain 4 of intermedilysin [rILYd4]), a 114 aa protein that inhibits human CD59 function with high affinity and specificity. In the presence of rILYd4, HIV-1 virions derived from either cell lines or peripheral blood mononuclear cells of HIV-1-infected patients became highly sensitive to complement-mediated lysis activated by either anti-HIV-1 gp120 Abs or by viral infection-induced Abs present in the plasma of HIV-1-infected individuals. We also demonstrated that rILYd4 together with serum or plasma from HIV-1-infected patients as a source of anti-HIV-1 Abs and complement did not mediate complement-mediated lysis of either erythrocytes or peripheral blood mononuclear cells. These results indicate that rILYd4 may represent a novel therapeutic agent against HIV-1/AIDS.