Glycine receptors (GlyRs) are crucial for the balance of excitation and inhibition in the mammalian spinal cord and brain stem. They belong to the superfamily of Cys-loop receptors and form pentameric ligand-gated chloride channels consisting of postsynaptic αβ heteromers or α homomers located extrasynaptically and at presynaptic membranes. Presynaptic GlyRs trigger depolarizing chloride currents upon activation by glycine spill-over.Autoantibodies (aAbs) against GlyRs are associated with the autoimmune disease Stiff Person Syndrome but also frequently found in patients with focal epilepsy. Loss of glycinergic function causes enhanced excitability leading to patients’ symptoms. At the molecular level, GlyR aAbs impair receptor function upon autoantibody binding and decrease surface expression upon receptor crosslinking and subsequent internalisation. So far, the findings on the pathophysiology of the aAbs concentrated on postsynaptic GlyR localization. However, aAbs targeting presynaptic GlyRs may also play a role in disease pathology.Here, super-resolution lattice SIM2 microscopy of ten patient serum samples revealed that aAbs not only target post- but also presynaptic GlyRs. Spinal cord interneuron (IN) cultures helped to specifically investigate the presynaptic component of glycinergic synapses. Analysis of these IN cultures via immunocytochemical stainings suggest the α2 subunit as target of GlyR aAbs at presynaptic sites. Incubation of mouse brainstem slices with patient sera caused a significant reduction in the frequency of spontaneous and miniature inhibitory postsynaptic currents emphasizing a contribution of altered presynaptic GlyR function to disease pathology.Overall, the investigation of additional molecular mechanisms could help to understand GlyR aAb pathology and identify novel therapeutic strategies.