Dihydroartemisinin (DHA), a natural product isolated from the traditional Chinese herb Artemisia annua and one of the clinical frontline drugs against malarial infections, has recently been discovered as a Toll-like Receptor 4 (TLR4) antagonist. However, the TLR4 antagonistic activity of DHA is modest and it exhibits cellular toxicity. In this work, the structure-activity relationship (SAR) of DHA as TLR4 antagonist was explored. Since destroying the sesquiterpene endoperoxide scaffold substantially compromised the TLR4 antagonistic activity and molecular dynamics analysis showed that the C-10 hydroxyl group formed a hydrogen bond with E72 of myeloid differentiation factor 2 (MD2) to prevent it moving deeper into MD2, SAR of DHA was focused on the C-10 hemiacetal position. With extending the length of the linear alkane chain at C10 position, the TLR4 antagonistic activity of DHA analogs increased first and then decreased with the best TLR4 antagonism occurring at the length of the carbon chain of 3-4 carbons. In contrast, the cellular toxicity of DHA analogs was raised with the increasing length of the linear alkane chain. The TLR4 antagonistic activity of DHA derivatives with substituted halogen as the terminal functional group decreased with the decrease of electronegativity of the substituted halogen, which implies the electron-rich functional group at the end of the alkane chain appears preferred. Therefore, DHA derivative 2k with alkynyl as the end functional group, exhibited 14 times more potent TLR4 antagonistic activity than DHA. Moreover, 2k showed less cellular toxicity than DHA. Cellular signaling characterizations indicated that 2k inhibited LPS-induced TLR4 dimerization and endocytosis and suppressed LPS-induced NF-κB but not MAPKs activation, culminating in blocking LPS-induced TLR4 signaling downstream pro-inflammatory factors NO and IL-1β. Further, 2k was active in vivo; it significantly increased and prolonged morphine analgesia. Collectively, this study provides a structural guidance to reposition DHA derivatives as TLR4 antagonists.