MicroRNA (miRNA) are a class of post‐transcriptional regulators of gene expression targeting mRNA for translational repression and/or degradation. We analyzed the expression of 365 miRNA in lymphocytes in relapsing–remitting MS patients, and show the first evidence for distinct miRNA expression profiles in CD4⁺, CD8⁺ and B cells in MS when compared with those in healthy volunteers. MiR‐17‐5p, which is involved in autoimmunity, was up‐regulated in CD4⁺ cells from MS patients. This was correlated with alterations in the expression of potential target genes of miR‐17‐5p, i.e. phosphatase and tensin homology and phosphatidyl‐inositol‐3‐kinase regulatory subunit 1, which were down‐regulated upon stimulation of CD4⁺ cells with anti‐CD3/CD28 in vitro. Functional experiments with a synthetic inhibitor of miR‐17 supported the link between miRNA expression and the altered target gene expression. Moreover, we found distinct responses of deregulated miRNA to stimulation, i.e. miR‐17‐5p and miR‐193a were strongly up‐regulated, in contrast to the down‐regulation of miR‐497, miR‐1 and miR‐126. Other deregulated miRNA did not respond to the stimulation probably due to other, non‐T‐cell activation related, mechanisms in their mode of action. Our findings support the role of miRNA‐dependent regulatory mechanisms in the immunopathogenesis of MS.