(+)-3a and (-)-3a were successfully separated from racemate (±)-3a by the chiral technique of supercritical fluid chromatography (SCF) with enantiomeric excess (ee%) >99% and purity >99%, and assigned for their absolute configuration as R and S, respectively, by the experimental electronic circular dichroism (ECD) spectrum and simulated ECD spectra calculated by time-dependent density functional theory (TDDFT) calculations. (+)-(R)-3a displayed excellent activity with an EC(50) of 5.3 nM against wild-type HIV-1, which was 12-fold more potent than (-)-(S)-3a. However, (-)-(S)-3a showed higher potency than (+)-(R)-3a against the double HIV-1 RT mutant (K103N+Y181C) as well as HIV-2 strain ROD. The possible reason for the difference of (R)- and (S)-3a in anti-HIV-1 activity was interpreted by molecular docking.