Abstract TLR7/9 signals are capable of mounting massive IFN response in pDCs immediately after viral infection, yet the involvement of epigenetic regulation in this process has not been documented. Through immune gene profiling, we found that the zinc finger CXXC family epigenetic regulator CXXC5 is specifically expressed in pDCs, but not in cDCs or macrophages. Interestingly, CXXC5-deficient pDCs exhibited impaired IFN-I response to TLR7/9 ligands and RNA/DNA viruses. Furthermore, we uncovered that CXXC5-deficiency resulted in aberrant methylation of CpG DNA islands (CGIs) of a subset of genes including Irf7 in the steady-state pDCs, leading to diminished expression of this group of genes in pDCs. Mechanistically, CXXC5 seemed to be responsible for the recruitment of DNA demethylase Tet2 to maintain the hypomethylation of these CGIs in pDCs, a process coincided with histone remodeling and active transcription in these regions. Upon challenged by viruses, CXXC5-deficient mice were unable to launch robust IFN-I response at the early stage, and became highly susceptible to HSV and VSV infection. Together, our results not only identified CXXC5 as a novel regulator of TLR7/9-induced IFN-I response in host defense, but also provided new insight into the understanding of epigenetic regulation of pDC function.