The intrathecal oligoclonal expansion of plasma cells in multiple sclerosis (MS) supports central nervous system (CNS) injury and is associated with active demyelination. The structure of the N-glycan at the conserved asparagine 297 in each of the CH2 domains of the Fc region of immunoglobulin G (IgG) directly affects its effector functions. In inflammation, IgG N-glycosylation patterns change and modulate pro-inflammatory or antiinflammatory effector functions, potentially reflecting MS status and activity. The aim of the research was to analyse N-glycosylation of serum and cerebrospinal fluid (CSF) IgG and total serum proteins in people with MS. A cross-sectional study included consecutive patients with suspected demyelinating disease of the CNS. The diagnosis of MS was based on the 2017 McDonald criteria, and the control group consisted of people in whom the diagnosis of MS was excluded. The analysis of fluorescently labelled N-glycans isolated from total serum proteins, serum and CSF IgG was performed by ultrahigh performance liquid chromatography based on hydrophilic interactions (HILIC-UPLC). N-glycans were compared with Expanded Disability Status Score (EDSS), signs of disease activity on magnetic resonance imaging (MRI), number of relapses and markers of intrathecal inflammation (cell count, IgG concentration in the CSF and percentage of intrathecal synthesis, oligoclonal bands, positive index specific antibodies to either measles, rubella, varicella zoster or herpes simplex viruses type 1 and 2 (MRZH reaction)). The differences between groups were found only in the N-glycome of CSF IgG. People with MS had a higher proportion of N-glycans with bisecting N-acetylglucosamine (P = 2.63 x 10-5) and monogalactosylated N-glycans (P = 1.49 x 10-6) which were associated with the presence of oligoclonal bands. The N-glycosylation properties, specifically structures monogalactosylated at the α6 antenna: FA2[6]G1 (r = 0.56, P < 0.001) and FA2[6]BG1 (r = 0.45, P = 0.001), correlated only with the percentage intrathecal synthesis of IgG, but not with the total concentration of IgG in the CSF. These structures were also more prevalent in MRZH positive people with MS. The FA2[6]BG1 structure was elevated in MS subjects with higher MRI lesion load (P = 0.0186), but CSF IgG Nglycosylation was not altered depending on the presence of active lesions, EDSS nor the number of relapses. Changes in N-glycosylation resulting in higher monogalactosylation of intrathecally synthesized IgGs are the most prominent features in MS and are associated with the radiological activity of the disease.