GB virus type C (GBV-C) is a common human flavivirus that has been associated with prolonged survival in HIV-positive individuals in several, though not all, epidemiological studies. There are five distinct GBV-C genotypes that are geographically localized, and it has been speculated that GBV-C genotypic differences may explain variable outcomes observed in different clinical studies. Expression of an 85 aa fragment of the GBV-C NS5A phosphoprotein (genotype 2) in a CD4+ T cell line (Jurkat) resulted in inhibition of HIV replication, mediated in part by decreased surface expression of the HIV coreceptor CXCR4 and upregulation of SDF-1. We expressed the NS5A protein from genotypes 1, 2, 3 and 5 in Jurkat cells, and demonstrated that all genotypes inhibited HIV replication. Further deletion mapping demonstrated that expression of a 30 aa fragment resulted in decreased CXCR4 surface expression, upregulation of SDF-1 and inhibition of HIV replication.