Subspecies of Trypanosoma brucei cause severe brain diseases after penetration of the blood-brain barrier. We investigated whether cytokines that modulate inflammatory cell infiltration into the brain also influence T. brucei neuroinvasion. Migration of a rodent pathogenic T. brucei strain from the cerebral blood vessels into the brain parenchyma was impeded in IFN-γ^–/–, IFN-γ receptor^–/– (IFN-γR^–/–), IL-12p40^–/–, and recombinant activating gene–1^–/– (RAG-1^–/–) mice as compared with their WT littermates despite higher levels of parasitemia in the mutant strains. Parasites accumulated in the perivascular compartment, confined between the endothelial and the parenchymal basement membranes, in certain areas of the brains of IFN-γ^–/–, IFN-γR^–/–, and RAG-1^–/– mice. This accumulation occurred around endothelial basement membranes containing the laminin α4 chain, while blood vessels showing robust laminin α5 chain immunostaining were not associated with parasite infiltration. The number of CD4⁺ and CD8⁺ T cells infiltrating the brain parenchyma was also reduced in the IFN-γ^–/– and IFN-γR^–/– mice. Our findings suggest that lymphocyte-derived IFN-γ facilitates trypanosome penetration across cerebral blood vessels and that the site of penetration is determined by the composition of the basement membranes of these vessels.