Mycoplasma pneumoniae (Mp) triggers Guillain-Barré syndrome (GBS) and elicits anti-galactocerebroside (GalC) antibodies. Specifically anti-GalC IgG is associated with Mp-GBS, possibly because of its better ability to cross the blood-nerve barrier (BNB). We here investigated CSF for the presence of anti-GalC in GBS. Intrathecal anti-GalC was found in 46% of Mp-GBS patients (n=6/13), in contrast to 16% of GBS controls (n=4/25) and 0% of non-GBS controls (n=0/7). The antibodies most likely originated from increased BNB permeability and/or intrathecal synthesis. Intrathecal anti-GalC IgG was specifically associated with Mp-GBS, further supporting that anti-GalC IgG contributes to the pathogenesis of GBS.