Two distinct types of CpG oligodeoxynucleotides have been identified that differ in their capacity to stimulate plasmacytoid dendritic cells (PDC): CpG‐A, but not CpG‐B, induces high amounts of IFN‐α, whereas CpG‐B induces PDC maturation. Here, we demonstrate that upon the inhibition of NF‐κB activation by SN50 or NBD, CpG‐B induced high amounts of IFN‐α, while CpG‐A‐induced IFN‐α was decreased. In addition, CpG‐B‐induced maturation was inhibited by NF‐κB inhibition. Using the new ImageStream imaging flow cytometry technology, we found that CpG‐B has a stronger ability to induce NF‐κB translocation in PDC than CpG‐A. Although both CpG‐A and CpG‐B‐induced NF‐κB translocation were inhibited by SN50, the inhibitory effect on CpG‐B‐induced translocation was more pronounced than with CpG‐A. Given this novel role for NF‐κB signaling in IFN‐α production, we tested whether there is a correlation between NF‐κB and IFN‐regulatory factor 7 (IRF‐7), the master regulator of IFN‐α production, activation. In contrast to the inability of CpG‐B to induce IRF‐7 translocation, we found that CpG‐A‐induced IRF‐7 translocation occurred at 5–6 hrs after stimulation. However, with the inhibition of NF‐κB translocation, CpG‐B induced high levels of IRF‐7 translocation after 1hr. These results suggest that NF‐κB plays an inverse role in the regulation of IFN‐α production by PDC upon stimulation with CpG‐A or CpG‐B.Supported by AI26806.