Inhibiting the α₄ subunit of the integrin heterodimers α₄β₁ and α₄β₇ with the monoclonal antibody natalizumab is an effective treatment for multiple sclerosis (MS). However, the pharmacological action of natalizumab is not understood conclusively. Previous studies suggested that natalizumab inhibits activation, proliferation, or extravasation of inflammatory cells. To specify which mechanisms, cell types, and α₄ heterodimers are affected by the antibody treatment, we studied MS-like experimental autoimmune encephalomyelitis (EAE) in mice lacking the β₁-integrin gene either in all hematopoietic cells or selectively in T lymphocytes. Our results show that T cells critically rely on β₁ integrins to accumulate in the central nervous system (CNS) during EAE, whereas CNS infiltration of β₁-deficient myeloid cells remains unaffected, suggesting that T cells are the main target of anti-α₄-antibody blockade. We demonstrate that β₁-integrin expression on encephalitogenic T cells is critical for EAE development, and we therefore exclude α₄β₇ as a target integrin of the antibody treatment. T cells lacking β₁ integrin are unable to firmly adhere to CNS endothelium in vivo, whereas their priming and expansion remain unaffected. Collectively, these results suggest that the primary action of natalizumab is interference with T cell extravasation via inhibition of α₄β₁ integrins.