The CCR5 co-receptor is necessary for cellular entry by R5 tropic viral strains involved in primary HIV infection, but is dispensable for normal human physiology. Owing to its crucial role in HIV-1 infection, the CCR5 co-receptor has been the subject of many therapeutic approaches, including gene therapy. siRNA targeting was shown to be effective in downregulating CCR5 expression and conferring significant protection against HIV-1 in susceptible cells. However, complete knockdown of CCR5 expression has not been achieved and thus remains an elusive goal. In these studies, we identified new CCR5 siRNAs capable of achieving complete knockdown of the co-receptor expression. Our transfection studies have shown that longer 28-mer short hairpin siRNAs are very effective in gene downregulation as assessed by fluorescence-activated cell sorting and transcript quantitation by quantitative real-time polymerase chain reaction. These siRNAs conferred strong antiviral protection during viral challenge. To obtain stable expression, highly potent siRNA expression cassettes were introduced into lentiviral vectors. Similar high levels of CCR5 downregulation were observed in stably transduced cells with concomitant viral protection in cultured cell lines. To translate these results to a stem cell gene therapy setting, CD34 hematopoietic progenitor cells were transduced with lentiviral vectors to derive transgenic macrophages. The transgenic cells also exhibited high levels of CCR5 downregulation and viral resistance. With regard to Pol-III promoter-mediated siRNA expression, higher efficacies were obtained with U6-driven CCR5 siRNAs. However, in contrast to previous reports, no apparent cytotoxicities were observed in transgenic cells containing U6-driven siRNA constructs. Thus the above anti-CCR5 siRNAs are among the most effective demonstrated to date and are very promising candidates for clinical applications.