Background: Dimethyl fumarate (DMF) alters the phenotype of circulating immune cells and causes lymphopenia in a subpopulation of treated multiple sclerosis (MS) patients. Objective: To phenotypically characterize circulating leukocytes in DMF-treated MS patients. Methods: Cross-sectional observational comparisons of peripheral blood from DMF-treated MS patients ( n = 17 lymphopenic and n = 24 non-lymphopenic), untreated MS patients ( n = 17) and healthy controls ( n = 23); immunophenotyped using flow cytometry. Longitudinal samples were analyzed for 13 DMF-treated patients. Results: Lymphopenic DMF-treated patients had significantly fewer circulating CD8⁺ and CD4⁺ T cells, CD56^dim natural killer (NK) cells, CD19⁺ B cells and plasmacytoid dendritic cells when compared to controls. CXCR3⁺ and CCR6⁺ expression was disproportionately reduced among CD4⁺ T cells, while the proportion of T-regulatory (T-reg) cells was unchanged. DMF did not affect circulating CD56^hi NKcells, monocytes or myeloid dendritic cells. Whether lymphopenic or not, DMF-treated patients had a lower proportion of circulating central and effector memory T cells and concomitant expansion of naïve T cells compared to the controls. Conclusions: DMF shifts the immunophenotypes of circulating T cells, causing a reduction of memory cells and a relative expansion of naïve cells, regardless of the absolute lymphocyte count. This may represent one mechanism of action of the drug. Lymphopenic patients had a disproportionate loss of CD8⁺ T-cells, which may affect their immunocompetence.