Mannan-binding lectin (MBL) belongs to the collectin family and functions as an opsonin that can also initiate complement activation. Our previous study showed that MBL serves as a double-stranded RNA binding protein that attenuates polyriboinosinic-polyribocytidylic acid-induced TLR3 activation. Prompted by these findings, in the present study cross-talk between MBL and CpG-DNA-induced TLR9 activation was investigated. Here, it was found that MBL also interacts with the TLR9 agonist, CpG oligodeoxynucleotide (CpG-ODN), in a calcium-dependent manner. Purified MBL protein suppressed activation of nuclear factor-kappa B signaling and subsequent production of proinflammatory cytokines from human monocytes induced by CpG-ODN 2006. These observations indicate that MBL can down-regulate CpG DNA-induced TLR9 activation, emphasizing the importance of understanding the interaction of MBL with TLR agonist in host immune defense.