Background Treatment with fingolimod reduces inflammation in multiple sclerosis (MS) by inhibiting lymphocyte egress from lymph nodes. We aimed to map, in detail, the alterations in peripheral blood lymphocyte subpopulations in relation to clinical outcome in MS patients treated with fingolimod. Methods Paired blood samples from relapsing-remitting MS patients (n = 19) were collected before and after one year of treatment with fingolimod (0.5 mg/day). Absolute counts and relative proportions of a broad set of T- B- and NK-cell subsets were analyzed by flow cytometry. Blood samples from 18 healthy controls were used for baseline comparisons. Results Treatment with fingolimod markedly decreased the absolute numbers of all major lymphocyte subsets, except for NK cells. The reduction was most pronounced within the T helper (Th) and B cell populations (p < 0.001). By phenotyping differentiation status of T cells, dramatic reductions within the naive and central memory (CM) cell populations were found (p < 0.001), while a less pronounced reduction was observed among effector memory (EM) cells (p < 0.001). The numbers of regulatory T cells (Tregs) were also decreased (p < 0.001), but to a lesser extent than other T cell populations, resulting in a relative preservation of Tregs with a memory phenotype (p = 0.002). Conclusions Our results confirm that fingolimod therapy markedly reduces lymphocyte counts in peripheral blood of MS patients. Subgroup analysis of T cells showed that naive and CM Th cells were the most profoundly affected and that memory Tregs were relatively preserved.