Transducing macrophages and other phagocytic cells has been problematic because these cells are largely nondividing and can phagocytose and degrade viral gene delivery vectors. Because of their carriage of the CCR5 chemokine receptor that functions as a coreceptor for most clinical strains of HIV, these cells are also key targets in early HIV infection and dissemination. We describe here a strategy to transduce these phagocytes, reduce cell membrane CCR5, and protect from infection with R5-tropic HIV. Recombinant Tag-deleted SV40 vectors were used to transduce unselected CCR5-bearing cell lines and primary cells with >98% efficiency. rSV40s were designed to express two different anti-CCR5 small interfering RNAs (siRNAs), driven by the adenoviral VA1 polymerase III (pol III) promoter, which localizes the transcripts in the cytoplasm. Transduction with both siRNAs substantially reduced CCR5 mRNA, which in turn decreased detectable cell membrane CCR5. Both CCR5+ cell lines and primary cells were used: SupT1/CCR5 cells, monocyte-derived macrophages (MDM), and primary human brain microglia. In addition, one siRNA, siRNA R5 #5, was designed to recognize conserved sequences in both murine and human CCR5 mRNA and effectively reduced CCR5 transcript in cells of both species. These siRNAs largely protected CCR5+ cell lines and primary human macrophages and brain microglia from challenge with R5-tropic HIV. Therefore, strategies to target CCR5 using rSV40-delivered, VA promoter-driven siRNAs may be useful therapeutic options for treating HIV infection.