Some α-helical antimicrobial peptides enhance the activation of immune cells induced by the recognition of DNA containing unmethylated cytosine-guanine motifs (CpG DNA). We recently found that an α-helical antimicrobial peptide FIKRIARLLRKIF, known as Kn2-7, increased CpG DNA-dependent responses in mouse macrophage- like RAW264.7 cells, and we also found that enhanced cellular uptake of CpG DNA by Kn2-7 was necessary but insufficient to augment CpG DNA-dependent responses. In this study, we clarified the relationship between the affinity of Kn2-7 to CpG DNA and the ability of Kn2-7 to enhance cellular uptake of DNA. Electrophoretic mobility analysis on a polyacrylamide gel revealed that Kn2-7 binds to CpG DNA more effectively than Kn2-7RA in which arginine residues of Kn2-7 were substituted with alanine residues, and also found that Kn2-7 binds to CpG DNA less effectively than Kn2-7KR in which lysine residues of Kn2-7 were substituted with arginine residues. The DNA-binding abilities of Kn2-7, Kn2-7RA, and Kn2-7KR correlated well with their abilities to enhance the cellular uptake of CpG DNA. In contrast, Kn2-7LA in which leucine residues of Kn2-7 were substituted with alanine residues exhibited a similar DNA-binding ability to Kn2-7, but it did not enhance cellular uptake of CpG DNA. Our results indicate that affinity to DNA is necessary for the ability of Kn2-7 to enhance cellular uptake of CpG DNA, but hydrophobicity of Kn2-7 is also necessary for the enhancement of cellular uptake.