We describe in this paper that the chloroxoquinolinic ribonucleoside 6-chloro-1,4-dihydro-4-oxo-1-(β-Dribofuranosyl)- quinoline-3-carboxylic acid (compound A) inhibits the HIV-1 replication in human primary cells. We initially observed that compound A inhibited HIV-1 infection in peripheral blood mononuclear cells (PBMCs) in a dosedependent manner, resulting in an EC50 of 1.5 ± 0.5 μM and in a selective index of 1134. Likewise, compound A blocked HIV-1BA-L replication in macrophages in a dose-dependent manner, with an EC50 equal to 4.98 ± 0.9 μM. The replication of HIV-1 isolates from subtypes C and F was also inhibited by compound A with the same efficiency. Compound A inhibited an early event of the HIV-1 replicative cycle, since it prevented viral DNA synthesis in PBMCs exposed to HIV-1. Kinetic assays demonstrated that compound A inhibits the HIV-1 enzyme reverse transcriptase (RT) in dose-dependent manner, with a KI equal to 0.5 ± 0.04 μM. Using a panel of HIV-1 isolates harboring NNRTI resistance mutations, we found a low degree of cross-resistance between compound A and clinical available NNRTIs. In addition, compound A exhibited additive effects with the RT inhibitors AZT and nevirapine, and synergized with the protease inhibitor atazanavir. Our results encourage continuous studies about the kinetic impact of compound A towards different catalytic forms of RT enzyme, and suggest that our nucleoside represents a promising molecule for future antiretroviral drug design.