Anti-interferon (IFN)–γ autoantibodies (AIGAs) are a pathogenic factor in late-onset immunodeficiency with disseminated mycobacterial and other opportunistic infections. AIGAs block IFN-γ function, but their effects on IFN-γ signaling are unknown. Using a single-cell capture method, we isolated 19 IFN-γ–reactive monoclonal antibodies (mAbs) from patients with AIGAs. All displayed high-affinity (K_D < 10⁻⁹ M) binding to IFN-γ, but only eight neutralized IFN-γ–STAT1 signaling and HLA-DR expression. Signal blockade and binding affinity were correlated and attributed to somatic hypermutations. Cross-competition assays identified three nonoverlapping binding sites (I–III) for AIGAs on IFN-γ. We found that site I mAb neutralized IFN-γ by blocking its binding to IFN-γR1. Site II and III mAbs bound the receptor-bound IFN-γ on the cell surface, abolishing IFN-γR1–IFN-γR2 heterodimerization and preventing downstream signaling. Site III mAbs mediated antibody-dependent cellular cytotoxicity, probably through antibody–IFN-γ complexes on cells. Pathogenic AIGAs underlie mycobacterial infections by the dual blockade of IFN-γ signaling and by eliminating IFN-γ–responsive cells.