To measure the prevalence of JCV-specific antibodies in a French cohort of MS patients treated with natalizumab and to identify risk factor(s) of JCV seropositivity. Progressive multifocal leukoencephalopathy (PML) risk may be stratified by anti-JCV antibody status, duration of natalizumab therapy (≥24 months) and prior exposure to immunosuppressive (IS) drugs. No data are available in France on the prevalence of anti-JCV antibodies and distribution of PML risk factors in patients treated with natalizumab. Sera of 361 patients under natalizumab therapy in two MS centers were analyzed using a previously validated ELISA test. We studied different characteristics: demographic, ethnic, radiological, clinical, prior use of immunomodulatory (IM) or IS drugs and natalizumab exposure duration. The JCV seropositivity rate was 51 % for the whole cohort. Mean natalizumab exposure duration was 27.27 months ± 15.57 (mean ± SD), and prior use of IS drugs was observed in 15.24 % of patients. Twenty-three patients (6.4 %) presented the three PML risk factors. By multivariate analysis, presence of anti-JCV antibodies was significantly linked to age, North African origin and natalizumab exposure duration. Anti-JCV antibody prevalence was similar to previously published data. Anti-JCV antibody status was linked to age. We also suggested that anti-JCV antibody status could be linked to natalizumab exposure duration and ethnic characteristics.