Summary Aims Fingolimod, an orally active immunomodulatory drug for relapsing‐remitting multiple sclerosis (RRMS), sequesters T cells in lymph nodes through functional antagonism of the sphingosine‐1‐phosphate receptor, reducing the number of potential autoreactive cells that migrate to the central nervous system. However, not all RRMS patients respond to this therapy. Our aim was to test the hypothesis that by immune‐monitoring RRMS patient's leukocyte subpopulations it is possible to find biomarkers associated with clinical response to fingolimod. Methods Prospective study. Analysis of peripheral blood mononuclear cell subpopulations by multiparametric flow cytometry, at baseline and +1, +3, +6, +12 months of follow‐up in 40 RRMS patients starting fingolimod therapy. Results Fingolimod treatment induced a severe lymphopenia affecting mainly T and B cells. A relative increase in Treg (memory Treg: 3.8 ± 1.0% baseline vs 8.8 ± 4.4% month +1; activated Treg: 1.5 ± 0.7% baseline vs 3.7 ± 2.1% month +1, P < 0.001) as well as transitional B cells (10.5 ± 12.3% baseline vs 18.7 ± 14.6% month +1, P < 0.001) was observed. Interestingly, lymphocyte subpopulations were already at baseline significantly different in responder patients. The percentage of recent thymic emigrants (RTE) used to stratify fingolimod responder, and no responder patients was the best biomarker (4.0 ± 1.4% vs 7.4 ± 1.9%, respectively P < 0.001). Conclusion The results support that immune‐monitoring of lymphocyte subpopulations in peripheral blood is a promising tool to select RRMS candidate for fingolimod treatment.