Human immunodeficiency virus type 1 (HIV-1) drug resistance and the latent reservoir are the two major obstacles to effectively controlling and curing HIV-1 infection. Therefore, it is critical to develop therapeutic strategies specifically targeting these two obstacles. Recently, we described a novel anti-HIV approach based on a modified human intrinsic restriction factor, R88-APOBEC3G (R88-A3G). In this study, we further characterized the antiviral potential of R88-A3GD128K (R88-A3Gm) against drug-resistant strains of HIV-1 and viruses produced from latently infected cells. We delivered R88-A3Gm into target cells using a doxycycline (Dox)-inducible lentiviral vector and demonstrated that its expression and antiviral activity were highly regulated by Dox. In the presence of Dox, R88-A3Gm-transduced T cells were resistant to infection caused by wild-type and various drug-resistant strains of HIV-1. Moreover, when the R88-A3Gm-expressing vector was transduced into the HIV-1 latently infected ACH-2 cell line or human CD4(+) T cells, on activation by phorbol-12-myristate-13-acetate or phytohemaglutinin, R88-A3Gm was able to curtail the replication of progeny viruses. Altogether, these data clearly indicate that R88-A3Gm is a highly potent HIV-1 inhibitor, and R88-A3Gm-based anti-HIV gene therapy is capable of targeting both active and latent HIV-1-infected cells to prevent subsequent viral replication and dissemination.Molecular Therapy-Nucleic Acids (2014) 3, e151; doi:10.1038/mtna.2014.2; published online 4 March 2014.