LL-37 is a cationic antimicrobial peptide derived from neutrophils and keratinocytes. It plays an important role in protection against bacterial infection in the skin and mucosal surfaces. However, its role within the blood compartment remains unclear given that serum inhibits its bactericidal property. In this study, we show that LL-37 promotes very rapid and highly efficient sensing of CpG motifs in bacterial DNA by human B lymphocytes and plasmacytoid dendritic cells (pDCs) in serum-containing media and in whole blood. LL-37 allowed detection of CpG oligodeoxynucleotide (ODN) within minutes of exposure. Without LL-37, 20-30 times more CpG was required to produce the same effect. The promotion of CpG detection by LL-37 was independent of the backbone of the ODN, as the effect was observed not only in ODNs with modified phosphorothioate backbone, but also in ODNs with natural phosphodiester backbone, as found in genomic DNA. Unmethylated CpG motifs within the phosphodiester ODN and LL-37-mediated delivery are required for pDCs to respond. In keeping with the above, cells responded to CpG-rich bacterial DNA and LL-37, but not to human DNA and LL-37. The ability of LL-37 to enhance delivery of CpG to stimulate immune cells is independent of its amphipathic structure and its bactericidal property. LL-37 aids the delivery of CpG to B cells and pDCs, but not T cells. These findings are pertinent to rapid recognition of microbial DNA and are highly relevant to contemporary studies of CpG/TLR9 agonists in vaccines and cancer therapy.