Background: Dimethyl fumarate (DMF) is used to treat relapsing multiple sclerosis and causes lymphopenia in a subpopulation of treated individuals. Much remains to be learned about how the drug affects B- and T-lymphocytes. Objectives: To characterize changes in B- and T-cell phenotype and function induced by DMF and to investigate whether low absolute lymphocyte count (ALC) is associated with unique functional changes. Methods: Peripheral blood mononuclear cells (PBMCs) were collected from DMF-treated patients, untreated patients, and healthy controls. A subset of DMF-treated patients was lymphopenic (ALC < 800). Multiparametric flow cytometry was used to evaluate cellular phenotypes. Functional response to non-specific and viral peptide stimulation was assessed. Results: DMF reduced circulating memory B-cells regardless of ALC. Follicular T-helper cells (CD4⁺ CXCR5⁺) and mucosal invariant T-cells (CD8⁺ CD161⁺) were also reduced. DMF reduced T-cell production of pro-inflammatory cytokines in response to polyclonal (PMA/ionomycin) and viral peptide stimulation, regardless of ALC. No differences in activation-induced cell death or circulating progenitors were observed between lymphopenic and non-lymphopenic DMF-treated patients. Conclusion: These data implicate DMF-induced changes in lymphocytes as an important component of the drug’s efficacy and expand our understanding of the functional significance of DMF-induced lymphopenia.