The binding of autoantibodies (autoAbs) to interferon (IFN)-γ in people with mycobacterial diseases has become an emerging medical concern. Many patients display specific human leukocyte antigen (HLA) class II haplotypes, which suggests that a common T cell-dependent and B cell-dependent mechanism might underlie the production of specific anti-IFN-γ autoAbs. We show here that these autoAbs target a major epitope (amino acids 121-131, designated position (P)121-131) in a region crucial for IFN-γ receptor (IFN-γR) activation to impair IFN-γ-mediated activities. The amino acid sequence of this epitope is highly homologous to a stretch in the Noc2 protein of Aspergillus spp., which was cross-reactive with autoAbs from patients. Rats immunized with Aspergillus Noc2 developed antibodies that reacted with human IFN-γ. We generated an epitope-erased variant of IFN-γ (EE-IFN-γ), in which the major neutralizing epitope region was altered. The binding affinity of anti-IFN-γ autoAbs for EE-IFN-γ was reduced by about 40%, as compared to that for IFN-γ1-131. Moreover, EE-IFN-γ activated the IFN-γR downstream signaling pathway ex vivo, irrespectively of anti-IFN-γ autoAbs. In conclusion, we identified a common, crucial B cell epitope that bound to anti-IFN-γ autoAbs in patients, and we propose a molecular-mimicry model for autoAb development. In addition, treatment with EE-IFN-γ might be worth investigating in patients producing anti-IFN-γ autoAbs.