CD4⁺ CD25^high regulatory T cells (Tregs) of patients with relapsing‐remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single‐cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4⁺ CD25^high FOXP3⁺ T cells and lower FOXP3 protein expression per cell in RR‐MS patients than in SP‐MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)‐β‐treated RR‐MS patients showed restored numbers of FOXP3⁺ Tregs. Furthermore, a higher percentage of CD4⁺ CD25^high FOXP3⁺ Tregs in RR‐MS patients, as compared with controls and SP‐MS patients, expressed CD103 and CD49d, adhesion molecules involved in T‐cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27⁺ CD25^high CD4⁺ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR‐MS patients. Taken together, these data show aberrant FOXP3 expression at the single‐cell level correlated with Treg dysfunction in RR‐MS patients. Our results also suggest that Tregs accumulate in the CSF of RR‐MS patients, in an attempt to down‐regulate local inflammation in the central nervous system.