The properties of gapmer antisense oligonucleotide (ASO) flanked by deoxyribonucleic guanidine (DNG) were investigated for the potential application in antisense technology. DNG is a unique nucleotide analog which has a positively charged internucleotide guanidinium linkage instead of negatively charged phosphodiester backbone linkage. We prepared a gapmer ASO containing DNG units at both wings of the sequence and compared its properties with 2′,4′-BNA/LNA gapmer ASOs with phosphorothioate (PS) backbone. Although DNG gapmer showed no stabilizing effect on the duplex formation with target RNA, the DNG modification was found to be tolerant to exonuclease digestion. Furthermore, DNG gapmer can induce RNase H-mediated cleavage of target RNA molecule, a requisite property for the antisense strategy. Therefore, the DNG gapmer developed in this study could be an interesting and useful candidate for the development of potent ASOs.