Background &Aims: Most Crohn’s disease (CD) patients show seroreactivity against Mycobacterium avium paratuberculosis (MAP), suggesting a pathogenic role for this organism. Our aim was to seek amino acid similarities between MAP and intestinal proteins that, through molecular mimicry,could serve as targets for cross-reactive immunity in CD.Methods: Fifty-three peptides comprising 23 sets of MAP/human intestinal peptidyl mimics chosen for maximal homology were constructed and tested for immunologic cross-reactivity by enzyme-linked immunosorbent assay in 50 patients with CD, 50 with ulcerative colitis, and 38 healthy controls. Results: Antibody reactivity was present in only 7 of 23 peptide sets. MAP/self-reactivity in at least 1 of the 7 reactive sets was present in 21 (42%) CD patients but was virtually absent in the controls. Significant double-reactivity was found against MAP glycosyl transferase d (gsd)230-244/human gastrointestinal glutathione peroxidase (GPg)111-125 homologues in 15 of 50 (30%) CD patients; MAP alkyl-ohydroperoxidase C (ahpC)20-34/human tumor overexpressed protein (TOG)637-651 double-reactivity was present in 10 (20%) CD patients, but in none of the controls. Inhibition studies confirmed that simultaneous reactivity to mimics was caused by cross-reactivity. Three-dimensional modeling predicts GPg111-125 will be exposed in a solvent-accessible surface region of the protein compatible with antibody recognition. Antibody affinity was greater for the MAP mimics than for the self-sequences, suggesting that reactivity to the mycobacterial sequences precedes that against self-sequences. Conclusions: We describe MAP/self-mimics as targets of cross-reactive antibody responses characterizing patients with CD. Our findings indicate gastrointestinal glutathione peroxidase as a novel autoantigen in CD. Background & Aims: Most Crohn’s disease (CD) patients show seroreactivity against Mycobacterium avium paratuberculosis (MAP), suggesting a pathogenic role for this organism. Our aim was to seek amino acid similarities between MAP and intestinal proteins that, through molecular mimicry, could serve as targets for cross-reactive immunity in CD. Methods: Fifty-three peptides comprising 23 sets of MAP / human intestinal peptidyl mimics chosen for maximal homologies were constructed and tested for immunologic cross-reactivity by enzyme-linked immunosorbent assay in 50 patients with CD, 50 with ulcerative colitis, and 38 healthy controls. Results: Antibody reactivity was present in only 7 of 23 peptide sets. MAP / self-reactivity in at least 1 of the 7 reactive sets was present in 21 (42%) CD patients but was virtually absent in the controls. Significant double-reactivity was found against MAP glycosyl transferase d (gsd) 230-244 / human gastrointestinal glutathione peroxidase (GPg) 111-125 homologues in MAP of -hydroperoxidase C (ahpC) 20-34 / human tumor overexpressed protein (TOG) 637-651 double-reactivity was present in 10 (20%) of CD patients, but in none of of the controls. Inhibition studies confirmed that simultaneous reactivity to mimics was caused by cross-reactivity. Three-dimensional modeling predicts GPg111-125 will be exposed in a solvent-accessible surface region of the protein compatible with antibody recognition. MAP mimics than for the self-sequences, suggesting that reactivity to the mycobacterial sequences precedes that against self-sequences. Conclusions: We describe MAP / self-mimics as targets of cross-reactive antibody responses characterizing patients with CD. Our findings include gastrointestinal glutathione peroxidase as a novel autoantigen in CD.