We applied heteroduplex oligonucleotide (HDO) technology, which uses an oligonucleotide hybridized with a complementary strand, to efficiently deliver locked nucleic acid (LNA)-based splice-switching oligonucleotides (SSOs) to the nucleus. Using an in vitro assay involving cationic lipids, we revealed that HDO technology increased the exon-skipping activity of LNA-based SSOs. To assess the effect of heteroduplex SSOs (HDSSOs) on exon-skipping activity, we designed and evaluated various HDSSOs using a series of complementary oligonucleotides with different sugar chemistries (DNA, RNA, and LNA), linkages (phosphodiester; PO and phosphorothioate; PS linkages), and lengths. HDO with different complementary oligonucleotide designs demonstrated a variety of exon-skipping activities. Next, we investigated the intracellular behavior of HDOs, which seemed to affect their efficient exon-skipping activity. We found that HDO technology increased the uptake of both SSOs and complementary oligonucleotides into the nuclei. Additionally, a series of complementary oligonucleotides showed different intracellular stabilities, and complementary oligonucleotide design appears to be one of the key factors affecting efficient exon skipping. Finally, we examined the exon-skipping activity of HDSSOs in mdx mice and found that HDSSOs exhibited higher exon-skipping activity than single-stranded LNA-based SSOs in these mice under intramuscular injections.