AbstractCRX‐527 belongs to a new family of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4‐phosphates, which are considered as potential vaccine adjuvants or stand‐alone immunotherapeutics to harness innate immune defenses. Since natural lipid A from bacterial LPS depends on membrane‐bound (mCD14) or soluble CD14 for its TLR4 ligand activity, we investigated the involvement of both forms of CD14 in the responses elicited by CRX‐527. First, we found that CRX‐527 induces NF‐κB and interferon regulatory factor‐3 (IRF‐3) activation in human embryonic kidney cells transfected with TLR4 and MD‐2 genes alone, whereas the responses to LPS require either co‐transfection of the gene encoding mCD14 or addition of soluble CD14. We then observed that monocyte‐derived DC, which are devoid of mCD14 respond to CRX‐527 but not to LPS in serum‐free medium. Furthermore, we found that, in contrast to LPS, CRX‐527 induces the production of cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which mCD14‐dependent responses are defective. Finally, we demonstrated that splenocytes from CD14‐deficient mice produce cytokines in response to CRX‐527 but not to LPS. We conclude that the lipid A mimetic CRX‐527 does not require the CD14 co‐receptor to elicit TLR4‐mediated responses.