Pdx1 is a key transcription factor involved in the regulation of insulin gene expression that is expressed at high levels in the β-cells of the pancreatic islets. We asked whether Pdx1 is a target of anti-islet autoimmunity in Type 1 diabetes (T1D). Pdx1 autoantibodies (PAA) were detected in non-obese diabetic (NOD) mice using ELISA, Western blotting, and radioimmunoprecipitation of [35S]-labeled insulinoma cell line-derived Pdx1 protein. PAA were detected as early as at 5 weeks of age, and generally peaked before the onset of clinically overt diabetes in diabetes-prone female NOD mice. Levels declined substantially after diabetes onset. PAA were not detected in the sera of NOD-scid, C57BL/6 or BALB/c mice. The titers of PAA in NOD mouse sera were as high as 1/93750 by ELISA. The fine specificity of PAA was determined by Western blotting using a series of truncated recombinant Pdx1 proteins. The immunodominant epitopes were located to the Pdx1 C-terminus (p200-283) in NOD mice. PAA also were detected in sera from human T1D patients, but the major epitopes were localized to amino acids 159-200 as well as the same region (p200-283) recognized by PAA from NOD mice. Using [3H]-thymidine incorporation, the p83 fragment of Pdx1 specifically stimulated proliferation of splenic T-cells from recent-onset diabetic NOD mice. The presence of PAA in prediabetic NOD mice and human T1D patients and Pdx1-specific T-cell proliferation in NOD mice provide a strong rationale for further investigation of the pathogenic role of immune responses against Pdx1 in T1D.