Inflammation, including reactive oxygen species and inflammatory cytokines in tissues amplify various post-translational modifications (PTMs) of self-proteins. A number of PTMs have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes (T1D). In this study, we have identified the citrullination of pancreatic glucokinase (GK) as a result of inflammation, triggering autoimmunity and affecting GK biological functions. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. We identify autoantibodies and autoreactive CD4+ T cells to glucokinase epitopes in the circulation of T1D patients and NOD mice. Finally, citrullination alters GK biologic activity (Km) and suppresses glucose-stimulated insulin secretion. Our studies define glucokinase as a T1D biomarker, providing new insights of how inflammation drives PTMs to create both neoautoantigens and affect beta cell metabolism.