In March and April 2009, an entirely novel influenza A (H1N1) virus (NIAV) emerged in Mexico and the USA. During the subsequent months, the virus rapidly spread all over the world by person-to-person transmission. In this report, RNA interference (RNAi) was used as an antiviral agent to inhibit NIAV replication in A549 cells. Ten small interfering RNAs (siRNAs) targeting extremely conserved regions among multiple NIAV genomes could effectively block the replication of NIAV strain A/Beijing/01/2009 (H1N1) in A549 cells. This study may be useful to confront the sudden emergence of NIAV infection.