We propose rational designing of antiviral short-interfering RNA (siRNA) targeting highly divergent HIV-1. In this study, conserved regions within HIV-1 genomes were identified through an exhaustive computational analysis, and the functionality of siRNAs targeting the highest possible conserved regions was validated. We present several promising antiviral siRNA candidates that effectively inhibited multiple subtypes of HIV-1 by targeting the best conserved regions in pandemic HIV-1 group M strains.