Secretory IgA (sIgA) antibodies in human tears and milk were found to recognize protein disulfide isomerase (PDI) on a Toxoplasma gondii lysate immunoblot (IB). These antibodies were already detectable in tears of infants. To determine the epitope containing-regions on PDI, we generated truncated versions of recombinant PDI that differ by 8-10 amino acids in length. By IB, it was found that the sIgA epitopes were confined to conserved regions of PDI, including the functionally essential thioredoxin-like domain. This suggested the capacity of sIgA to react with PDI of other species, which was confirmed by recognition of human PDI by IgA in tears. In contrast, anti-T. gondii PDI antibodies generated by immunization were not able to cross-react. Binding to the thioredoxin-like domain on IB could be gradually abrogated by incubation with peptide constituting the same domain. By consecutive investigation of the function of the protein targeted by sIgA, the presence of antibody in relation to age and analysis of the epitope constituting regions on PDI we demonstrate that sIgA directed against PDI are self-reactive natural antibodies. Furthermore, analysis of antibody epitopes on an antigen is a useful method to distinguish conventional, affinity-matured antibodies from natural antibodies. The presence at early age and continuity of anti-PDI sIgA in relation to age suggests the existence of B cells secreting germline-encoded antibodies in human mucosa outside of the gut. Overall, the PDI-specific antibodies are clearly part of the natural antibody repertoire, suggesting an active role for these antibodies in the innate defense against pathogens.