Dimethyl fumarate (DMF; trade name Tecfidera™) is an oral formulation of the fumaric acid ester that is FDA approved for treatment of relapsing-remitting multiple sclerosis (RRMS). To better understand the therapeutic effects of Tecfidera and its rare side-effect of progressive multifocal leukoencephalopathy (PML), we conducted cross-sectional and longitudinal studies by immunophenotyping cells from peripheral blood (particularly T lymphocytes) derived from untreated, 4-6 month and 18-26 month Tecfidera-treated stable RRMS patients using multi-parametric flow cytometry. The absolute numbers of CD4 and CD8 T cells were significantly decreased and the CD4/CD8 ratio was increased with DMF treatment. The proportion of both effector memory (Tem) and central memory T cells (Tcm) were reduced while naïve T cells (Tn) increased in treated patients. T cell activation was reduced with DMF treatment especially among Tem and effector memory RA (Temra) T cells. T helper subsets Th1 (CXCR3+), Th17 (CCR6+), and particularly those expressing both CXCR3 and CD161 were reduced most significantly, while the anti-inflammatory Th2 subset (CCR3+) was increased after DMF treatment. A corresponding increase in IL-4, and decrease in IFNγ and IL-17-expressing CD4+ T cells was observed in DMF-treated patients. DMF in vitro treatment also led to increased T cell apoptosis and decreased activation, proliferation, reactive oxygen species, and CCR7 expression. Our results suggest that DMF acts on specific memory and effector T cell subsets by limiting their survival, proliferation, activation, and cytokine production. Monitoring these subsets could help to evaluate the efficacy and safety of DMF treatment.