SIGNR1, a member of a new family of mouse C-type lectins, is expressed at high levels in macrophages (Mphi) within the splenic marginal zone, lymph node medulla, and in some strains, in peritoneal cavity. We previously reported that SIGNR1 captures gram-negative bacteria, such as Escherichia coli and Salmonella typhimurium, as well as Candida albicans. We have now investigated the precise ligands and innate responses that involve SIGNR1. The interaction of SIGNR1 with FITC-dextran and E. coli was completely inhibited by LPS from E. coli and Salmonella minnesota. Using LPS from various types of rough mutants of Salmonella, we found that SIGNR1 primarily recognizes oligosaccharides in the non-reductive end of the LPS core region. In transfectants, expression of SIGNR1 enhanced the oligomerization of Toll-like receptor (TLR) 4 molecules as well as the degradation of IkappaB-alpha after stimulation with E. coli under low-serum conditions. The enhanced TLR4 oligomerization was inhibited by pre-treatment of the cells with anti-SIGNR1 mAb or with mannan. A physical association between SIGNR1 and the TLR4-MD-2 complex was also observed by immunoprecipitation. Finally, we found that transfection of SIGNR1 into the macrophage-like RAW264.7 cells resulted in significant augmentation of cytokine production. These results suggest that SIGNR1 associates with TLR4 to capture gram-negative bacteria and facilitate signal transduction to activate innate M responses.