Recruitment of naturally occurring CD4⁺ CD25⁺ regulatory T (T_reg) cells is a highly promising approach for the treatment of experimental autoimmune encephalomyelitis (EAE), a widely used model of multiple sclerosis. Here, we studied the in vivo interaction of T_reg cells, induced by the monoclonal anti-CD28 antibody JJ316, with encephalitogenic T cell lines established from eGFP-transgenic rats. By tracking these fluorescent cells using flow cytometry and confocal microscopy, we found that the activation and expansion of T_reg cells inhibited infiltration of the CNS by pathogenic T cells. Interference with effector cell migration occured within the secondary lymphoid organs, since the early therapeutic effects were achieved despite the absence of T_reg cells in the spinal cord. However, the delayed homing to the CNS seen after prophylactic JJ316 administration indicates that T_reg cells may play an additional role within the target tissue. In addition, the blood–brain barrier remained largely intact after JJ316 treatment, the secretion of T_H2 cytokines was augmented and the encephalitogenic T cells exhibited a reduced secretion of IFN-γ. This in turn resulted in a reduced expression of the chemokine receptor CXCR-3 on effector T cells which may interfere with their capacity to infiltrate the CNS. Importantly, these effects were not achieved by direct action of JJ316 on the encephalitogenic cells. Our data rather suggest that polyclonal activation of T_reg cells in the secondary lymphoid organs is instrumental in preventing the pathological transmigration of encephalitogenic T cells into the CNS. We anticipate that these results may help to better understand the role of T_reg cells in controlling autoimmunity in the CNS.