Autoantibodies in Type 1 diabetes patients may differentiate between glutamate decarboxylase (GAD65) cloned from human, mouse and rat with a significant better binding to the human antigen. A subgroup of 15% (27/183) patients showed significantly better binding to rodent than to human GAD65. The aim of this study was to determine whether the autoantibody specificity would remain anti-rodent during longitudinal follow-up for one year. We observed 1) that the average slope of the difference between human and mouse GAD65 autoantibodies binding increased between onset and after one year, which demonstrates reduced binding to rodent GAD65 and 2) that, in a group followed every third month, 9/11 (80%) children with rodent specific GAD65 autoantibodies at onset converted within one year to preference against human GAD65. This shift in preference was confirmed by significantly lower EC50 values in the initially anti-rodent GAD65 autoantibodies compared to samples taken one year after clinical diagnosis as determined in displacement studies with unlabeled human GAD65. We speculate that the evolution of GAD65 autoantibodies in Type 1 diabetes includes reactivity to a non-human GAD65 N-terminal end conformation. Progression towards Type 1 diabetes is, however, associated with a maturation of the immune response towards human GAD65 autoreactivity.