<jats:sec><jats:title>Background and purpose</jats:title><jats:p>Cytomegalovirus (<jats:styled-content style="fixed-case">CMV</jats:styled-content>) infection has recently been associated with a lower multiple sclerosis (<jats:styled-content style="fixed-case">MS</jats:styled-content>) susceptibility, although it remains controversial whether it has a protective role or is merely an epiphenomenon related to westernization and early‐life viral infections. We aimed to evaluate whether <jats:styled-content style="fixed-case">CMV</jats:styled-content> serostatus may differ in patients with early <jats:styled-content style="fixed-case">MS</jats:styled-content> as compared with patients with non‐early <jats:styled-content style="fixed-case">MS</jats:styled-content>, analyzing the putative association of this virus with <jats:styled-content style="fixed-case">MS</jats:styled-content> clinical course and humoral immune responses against other herpesviruses.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Multicentric analysis was undertaken of 310 patients with <jats:styled-content style="fixed-case">MS</jats:styled-content> (early <jats:styled-content style="fixed-case">MS</jats:styled-content>, disease duration ≤5 years, <jats:italic>n</jats:italic> = 127) and controls (<jats:italic>n</jats:italic> = 155), evaluating specific humoral responses to <jats:styled-content style="fixed-case">CMV</jats:styled-content>, Epstein–Barr virus and human herpesvirus‐6, as well as T‐cell and natural killer (<jats:styled-content style="fixed-case">NK</jats:styled-content>)‐cell immunophenotypes.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Cytomegalovirus seroprevalence in early <jats:styled-content style="fixed-case">MS</jats:styled-content> was lower than in non‐early <jats:styled-content style="fixed-case">MS</jats:styled-content> or controls (<jats:italic>P</jats:italic> < 0.01), being independently associated with disease duration (odds ratio, 1.04; 95% confidence interval, 1.01–1.08, <jats:italic>P</jats:italic> < 0.05). <jats:styled-content style="fixed-case">CMV</jats:styled-content>+ patients with <jats:styled-content style="fixed-case">MS</jats:styled-content> displayed increased proportions of differentiated T‐cells (<jats:styled-content style="fixed-case">CD</jats:styled-content>27−<jats:styled-content style="fixed-case">CD</jats:styled-content>28−, <jats:styled-content style="fixed-case">CD</jats:styled-content>57+, <jats:styled-content style="fixed-case">LILRB</jats:styled-content>1+) and <jats:styled-content style="fixed-case">NKG</jats:styled-content>2C+ <jats:styled-content style="fixed-case">NK</jats:styled-content>‐cells, which were associated with a lower disability in early <jats:styled-content style="fixed-case">MS</jats:styled-content> (<jats:italic>P</jats:italic> < 0.05). <jats:styled-content style="fixed-case">CMV</jats:styled-content>+ patients with early <jats:styled-content style="fixed-case">MS</jats:styled-content> had an age‐related decline in serum anti‐<jats:styled-content style="fixed-case">EBNA</jats:styled-content>‐1 antibodies (<jats:italic>P</jats:italic> < 0.01), but no <jats:styled-content style="fixed-case">CMV</jats:styled-content>‐related differences in anti‐human herpesvirus‐6 humoral responses.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Low <jats:styled-content style="fixed-case">CMV</jats:styled-content> seroprevalence was observed in patients with early <jats:styled-content style="fixed-case">MS</jats:styled-content>. Modification of <jats:styled-content style="fixed-case">MS</jats:styled-content> risk attributed to <jats:styled-content style="fixed-case">CMV</jats:styled-content> might be related to the induction of differentiated T‐cell and <jats:styled-content style="fixed-case">NK</jats:styled-content>‐cell subsets and/or modulation of Epstein–Barr virus‐specific immune responses at early stages of the disease.</jats:p></jats:sec>