Follicular helper T (Tfh) cells and their interplay with B cells likely contribute to the pathogenesis of relapsing-remitting multiple sclerosis (RRMS). Tfh cells are enriched in cerebrospinal fluid (CSF) in RRMS, but effects of anti-CD20 therapy are unknown. We investigated Tfh cells in controls, untreated and anti-CD20-treated patients with RRMS using flow cytometry. CSF Tfh cells were increased in untreated patients. Compared to paired blood samples, CD25⁻ Tfh cells were enriched in CSF in RRMS, but not in controls. Contrast-enhancing brain MRI lesions and IgG index correlated with CSF CD25⁻ Tfh cell frequency in untreated patients with RRMS. Anti-CD20 therapy reduced the numbers of circulating PD1⁺ Tfh cells and CD25⁻ Tfh cells, and the frequency of CSF CD25⁻ Tfh cells. The study suggests that CD25⁻ Tfh cells are recruited to the CSF in RRMS, associated with focal inflammation, and are reduced by anti-CD20 therapy.