Objective To compare the effects of two approaches of SIVmac239 infection in endothelial cells, directly by cell-free virus or by T cell-mediated infection, to explore the predominant approach of SIVmac239 infection in endothelial cells, and finally lay a theoretical foundation of the molecular mechanism of SIV viral disruption of the blood-brain barrier. Methods We adopted two distinct ways to infect endothelial cells. One was to directly infect the endothelial cells by cell-free virus, another was by co-culture of infected CEMx174 cells with the endothelial cells. The degree of infection in the endothelial cells was evaluated by nested-PCR, indirect immunofluorescence assay, Western blotting and ELISA assays. Results The provirus DNA was found in the endothelial cells infected in either way. However, the endothelial cells infected by co-culture with CEMx174 cells had a significantly higher amount of provirus DNA and higher expression of SIV P27 in the cells and in the supernatant than those in the ECs directly infected by cell-free virus. Conclusions The way of T cell-mediated infection substantially enhances the capacity of SIV infection of endothelial cells than the direct infection by cell-free virus. It indicates that the T cell-mediated infection may serve as the principal route of SIV disruption of the blood-brain barrier.