The human zinc transporter Slc30A8 (ZnT8) is a major target of humoral autoimmunity in human type 1A diabetes. However, despite extensive conservation, the majority of human autoimmune sera fail to recognize the murine ortholog. Moreover, Slc30A8 appears not to be a significant target of humoral autoimmunity in the NOD mouse. We therefore “humanized” the murine protein by site-directed mutagenesis. Only conversion of Q324 to arginine (equivalent to R325 in the human protein) partially restored reactivity to a pool of sera selected for high titers to the human probe. Additionally, the reciprocal mutation (human R325 to Q) abolished reactivity for 38/103 (36.9%) of ZnT8+ sera. We conclude that the C-terminal domain of human ZnT8 contains at least two discrete epitopes, one of which is critically dependent upon the arginine residue at position 325.