Dendritic cells (DCs) are initiators of innate immunity and acquired immunity through the production of cytokines such as interferon-α (IFN-α) and interleukin-12 (IL-12). Nucleic acids (DNA, RNA) from damaged cells or pathogens are important activators not only for anti-microbial innate immune responses but also in the pathogenesis of autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the nucleic acids-mediated innate immunity by possessing toll-like receptor 9 (TLR9). We here found that double-stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte-derived DCs (monoDCs), leading to the preferential production of IFN-α but not IL-12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA-mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by monoDCs without co-localization of endosomal LAMP1 staining, and the dsDNA-mediated IFN-α production was not impaired by chloroquine. These findings indicate that monoDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single-stranded RNA (ssRNA) stimulated monoDCs to secrete IL-12 but not IFN-α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA-mediated monoDC activation. As might be inferred from our findings, monoDC may function as a traffic control between innate immunity via IFN-α production and acquired immunity via IL-12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of monoDC underlying the dsDNA-mediated activation of protective or pathogenic immunity.