Non-nucleoside reverse-transcriptase inhibitors (NNRTIs), major components of highly active antiretroviral therapy (HAART), are effective in suppressing viral replication and preventing the progress of HIV-1 infection to AIDS. However, rapid blood clearance in vivo could significantly impair the efficiency of the anti-HIV-1 activity and result in multiple daily doses which might lead to poor patient compliance. Here we attempted to employ biodegradable organic nanoparticles (NPs) to encapsulate DAAN15h, a derivative of 4-substituted 1, 5-diarylaniline with potent anti-HIV activities. Nanoparticles encapsulating DAAN15h (NP-DAAN15h) displayed a spherical shape with a size of 97.01 ± 3.64 nm and zeta potential of -19.1 ± 3.78 mV, and they exhibited a sustained controlled release behavior in vitro. The cellular uptake of NPs on TZM-b1 cells, MT-2 cells and M7 cells, possibly through lipid raft-mediated and energydependent active transport processes, was significantly enhanced. NP-DAAN15h, which possessed no significant in vitro cytotoxicity, showed improved antiviral activity against laboratory-adapted and primary HIV-1 isolates with different subtypes and tropisms, including RT-resistant variants. NP-DAAN15h exhibited a significantly prolonged blood circulation time, decreased plasma elimination rate, and enhanced AUC(0-t). NP-DAAN15h, a nanoparticle-encapsulated NNRTI, exhibits enhanced cellular uptake, improved anti-HIV-1 efficacy and prolonged in vivo circulation time, suggesting good potential for further development as a new NNRTI formulation for clinical use.