We investigated 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides (AONs) for vascular endothelial growth factor (VEGF) in human lung carcinoma A549 cells. An ENA/DNA gapmer AON with RNase H-mediated activity was virtually stable in rat plasma and exhibited more than 90% inhibition of VEGF mRNA production. Moreover, 22 genes that are likely to bind to the AON were found in the GenBank database by BLAST and CLUSTAL W searches. Three of these genes were actually inhibited by the ENA AON. In shorter ENA AONs with fewer matched sequences of these genes, inhibitiory activities were decreased and off-target effects were improved. These results indicate that ENA AONs act in a sequence-specific manner and could be used as effective antisense drugs.