Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), clinically characterized by alternating periods of relapse and remission and usually by a progressive paralysis. Whereas the cause of MS is multifactorial, a central role has been attributed to autoimmune responses against components of the myelin sheath coating the nerve cells of the CNS. The CNS myelin sheath contains proteins and is also rich in glycosphingolipids, particularly sulfatide and galactosylceramide. Sulfatide is a ligand for non-conventional T lymphocytes such as natural killer T (NKT) cells and TCRγδ T cells. Recently, sulfatide-reactive CD1d-restricted T cells in human peripheral blood were found to be TCRγδ T cells that preferentially use the TCR Vδ1-segment, a TCR previously identified in MS lesions. However, a putative role for Vδ1 T cells in immunopathogenesis of MS is poorly understood. In this study, we have investigated the TCR Vδ1 cells in peripheral blood and cerebrospinal fluid (CSF) of newly diagnosed MS patients using 14-color flow cytometry. Vδ1 T cells were present in CSF and displayed markers of recent activation, and the frequency of this subset was significantly increased in peripheral blood from patients as compared to healthy donors. Moreover, newly diagnosed MS patients had a dramatically increased frequency of IFN-γ producing Vδ1 T cells in peripheral blood compared to healthy donors. This contrasts to the anti-inflammatory profile normally associated with Vδ1 T cells. We found no change in the cytokine production in other T cell subsets analyzed. Notably, MS patients treated with Natalizumab (blocking integrin α-4 and thereby leukocyte homing to the CNS) had normalized IFN-γ levels in Vδ1 T cells. Taken together, this suggest that Vδ1 T cells are functionally altered early in MS, and may be involved in the early pathogenesis of the disease by producing proinflammatory cytokines.