A retroviral element (MSRV) defining a family of genetically inherited endogenous retroviruses (HERV-W) has recently been characterized in cell cultures from patients with multiple sclerosis (MS). To address the possible relationship with MS, direct detection of circulating virion RNA was proposed but revealed technically difficult to perform in standardized conditions, in the face of multiple endogenous HERV-W copies. A parallel approach has evaluated MSRV potential pathogenicity in relation to characteristic features of multiple sclerosis, in particular, T-lymphocyte-mediated immunopathology. We report here that MSRV particles induce T-lymphocyte response with a bias in the Vβ16 chain usage in surface receptor, whatever the HLA DR of the donor. A recombinant MSRV envelope—but not core—protein reproduced similar nonconventional activation. Molecular analysis of Vβ CDR3 showed that Vβ16 expansions are polyclonal. Our results thus provide evidence that MSRV envelope protein can trigger an abnormal immune response with similar characteristics to that of superantigens.