Angiogenin (ANG, also known as ribonuclease 5) is a 14.1kDa polypeptide secreted by T lymphocytes and epithelial cells. Other than its well-characterized function of promoting development of vasculature, ANG also possesses anti- Human Immunodeficiency Virus-1 (HIV-1) properties. Previous findings show that ANG inhibits X4 tropic HIV-1 replication in peripheral blood mononuclear cells (PBMCs). Our studies show that ANG also inhibits R5 tropic virus, the most commonly transmitted HIV-1 strain. Inhibition was detected treating cells prior to or after infection by p24 ELISA, and is not mediated by CD4, CXCR4, or CCR5 as determined by flow cytometry assays. Furthermore, we demonstrated that ANG binds to tRNA-Lys, which serves as primer for HIV-1 reverse transcription. These results indicate that ANG inhibits HIV-1 replication by a post-entry mechanism, which might be exploited to develop new antiretroviral strategies.