In order to evaluate the function of the mouse peptidylarginine deiminase 4 (PADI4) gene, gapmer-designed ENA antisense oligonucleotides were utilized. Antisense ENA gapmers were found to inhibit the mRNA expression of the PADI4 gene, but ENA gapmers with sense sequences as controls only partially inhibited this mRNA expression. An in vitro E. coli RNase H reaction analysis using transcripts of the PADI4 gene and their ENA gapmers revealed that the mixtures of the transcripts and ENA gapmers with sense sequences were cleaved at non-target sites by RNase H derived from partial base-pairing between the transcript and the ENA gapmer. In an in vitro E. coli RNase H reaction analysis, when the DNA region of the ENA gapmer was shortened to 5 or 6 nucleotides, the non-target cleavage disappeared and the specific inhibition of PADI4 mRNA expression was observed. These results demonstrated that ENA gapmers with a short DNA region improved the sequence-specificity of mRNA down-regulation. As well, they suggest that we should be alert to the utility of antisense oligonucleotides with a wide DNA region in terms of sequence specificity, and additionally, that optimized ENA gapmers could be useful for application to gene validation.