TLR4 is an important pattern recognition receptor that plays important roles in innate immunity against infections. CD317 is a type Ⅱ transmembrane glycoprotein with unique structure that endows it viral tethering and signaling potential. However, it is not clear yet whether CD317 regulates TLR4-mediated signal transduction. Herein, we investigated the role of CD317 on TLR4 signal transduction by using primary peritoneal macrophages derived from CD317 knockout mice and THP-1-derived human macrophages, and found that the expression of CD317 could be induced by TLR4 agonist (Lipopolysaccharide), which, in a feedback loop, further enhanced the TLR4-mediated NF-κB activation and subsequent release of pro inflammatory cytokines (TNF-α, IL-1β and IL-6, etc.). CD317 knockdown or knockout markedly impaired the TLR4 signal transduction and cytokine production. By using co-immunoprecipitation, we found that CD317 could interact with both MyD88 and TRAF6, suggesting that CD317 probably enhanced the TLR- 4-mediated NF-κB activation through MyD88-TRAF6 signal transduction pathway. Conclusively, our study not only extends the understanding on the role of CD317 in innate immune regulation, but also provides new targets and theoretical guidance for the development of effective strategies for related diseases.