Transplantation of ex vivo gene-corrected autologous cells represents an attractive therapeutic approach for brain diseases. Among the cells of the central nervous system, brain macrophages are promising candidates due to their role in tissue homeostasis and their implication in several neurological diseases. Up to now, gene transfer into macrophages has proven difficult by most currently available gene delivery methods. We describe herein, an efficient transduction of rat bone marrow-derived and brain macrophages with an HIV-1-derived vector containing a central DNA flap and encoding the GFP reporter gene (TRIP-DeltaU3-GFP). In primary cultures of macrophages our results show that more than 90% of the cells were transduced by the TRIP vector and that GFP expression remained stable for 1 month without cytopathic effect. In vivo, transplants of transduced macrophages into the striatum of adult rats exhibited long-term expression of GFP up to 3 months. Transduced macrophages were observed around the brain injection site and exhibited the brain macrophage/microglia phenotype. There was no significant sign of astrogliosis around the graft. These results confirm the potential of lentiviral vectors for efficient and stable ex vivo transduction of macrophages. Moreover, transduced autologous macrophages appear as a valuable vehicle for long-term and localized gene expression into the brain.