RNA interference (RNAi) has become an indispensable technology for biomedical research and has demonstrated the potential to become a new class of therapeutic. Current RNAi technology in mammalian cells relies on short interfering RNA (siRNA) consisting of symmetrical duplexes of 19-21 base pairs (bp) with 3' overhangs. Here we report that asymmetric RNA duplexes with 3' and 5' antisense overhangs silence mammalian genes effectively. An asymmetric interfering RNA (aiRNA) of 15 bp was incorporated into the RNA-induced silencing complex (RISC) and mediated sequence-specific cleavage of the target mRNA between base 10 and 11 relative to the 5' end of the antisense strand. The gene silencing mediated by aiRNA was efficacious, durable and correlated with reduced off-target silencing by the sense strand. These results establish aiRNA as a scaffold structure for designing RNA duplexes to induce RNAi in mammalian cells.