Toll-like receptor 4 (TLR4) is involved in CD4+ T lymphocyte-mediated pathologies. Here, we demonstrate that CD4+ T lymphocytes express functional TLR4 that contributes to their activation, proliferation and cytokine secretion. In addition, we demonstrate that TLR4-induced responses are mediated by macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine. We also demonstrate that MIF regulates suboptimal TCR/CD3-mediated activation of T lymphocytes. On one hand, MIF prevents excessive TCR/CD3-mediated activation of CD4+ T lymphocytes under suboptimal stimulation conditions and, on the other hand, MIF enables activated CD4+ T lymphocytes to sense their microenvironment and adapt their effector response through TLR4. Therefore, MIF appears to be a major regulator of the activation of CD4+ T lymphocytes and the intensity of their effector response. TLR4-mediated activation is thus an important process for T cell-mediated immunity.