Ninjurin1 is a transmembrane protein involved in macrophage migration and adhesion during inflammation. It was recently reported that repression of Ninjurin1 attenuated the lipopolysaccharide (LPS)-induced inflammatory response in macrophages; however, the precise mechanism by which Ninjurin1 modulates LPS-induced inflammation remains poorly understood. In the present study, we found that the interaction between Ninjurin1 and LPS contributed to the LPS-induced inflammatory response. Notably, pull-down assays using lysates from HEK293T cells transfected with human or mouse Ninjurin1 and biotinylated LPS (LPS-biotin) showed that LPS directly bound Ninjurin1. Subsequently, LPS binding assays with various truncated forms of Ninjurin1 protein revealed that amino acids (aa) 81-100 of Ninjurin1 were required for LPS binding. In addition, knockdown experiments using Ninj1 siRNA resulted in decreased nitric oxide (NO) and tumor necrosis factor-α (TNFα) secretion upon LPS treatment in Raw264.7 cells. Collectively, our results suggest that Ninjurin1 regulates the LPS-induced inflammatory response through its direct binding to LPS, thus, identifying Ninjurin1 as a putative target for the treatment of inflammatory diseases, such as sepsis and inflammation-associated carcinogenesis.