GAD65 antibodies target the 65 kDa isoform of glutamic acid decarboxylase (GAD), an enzyme responsible for converting glutamate to gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system. Primarily expressed in pancreatic β-cells and neuronal tissues, GAD65 plays a critical role in maintaining GABAergic signaling. Autoantibodies against GAD65 are strongly associated with autoimmune disorders, notably type 1 diabetes (T1D) and stiff-person syndrome (SPS). In T1D, these antibodies contribute to β-cell destruction, serving as a diagnostic marker for autoimmune-mediated diabetes. In SPS, a rare neurological disorder, GAD65 antibodies are linked to impaired GABAergic inhibition, leading to muscle rigidity and spasms. They are also observed in other neurological conditions, including cerebellar ataxia and epilepsy. Unlike GAD67 (the other GAD isoform), GAD65 is membrane-associated and regulated by ATP, reflecting its role in rapid GABA synthesis. Detection methods (e.g., ELISA, radioimmunoassay) aid in diagnosing autoimmune diseases, though antibody titers do not always correlate with disease severity. Research continues to explore their pathogenic mechanisms and therapeutic targeting.