Aim Antisense oligonucleotide (ASO) has the potential to induce off-target effects by inadvertent binding of ASOs to unintended RNAs that have a sequence similar to the target RNA. In the present study, we focused on the association between oligonucleotide length and off-target effects. Oligonucleotide extension is assumed to have bilateral effects on hybridization-dependent changes in gene expression, i.e., one is the decrease of off-target effects based on the reduced number of off-target candidate genes with perfect matches, and the other is the increase of off-target effects based on the increased binding affinity between the ASO and the complementary RNAs that leads to better tolerability for mismatches. Methods To determine the effects of oligonucleotide extension of gapmer ASOs on off-target effects, an extensive microarray analysis was performed using human cells treated with a 14-mer gapmer ASO and the extended 18-mer derivatives with the same core 14-mer region. Results and Discussion Our data indicated that change in gene expression in the cells treated with 18-mer ASOs was significantly smaller than those with a 14-mer ASO, showing the decrease of off-target effects by oligonucleotide extension.