Myeloid differentiation protein 2 (MD2), a key TLR4adaptor protein for sensing LPS, plays animportant role ininflammatoryprocessand has been identified as a promising target for the treatment of avarietyof inflammatory diseases. In our study, aseries of benzoxazolone derivatives weresynthesized, characterized and tested for anti-inflammatory activity in vitro. The compounds 3c, 3dand 3gdemonstrated the greatestanti-inflammatory activityagainst IL-6 with IC50values of 10.14±0.08, 5.43±0.51 and 5.09±0.88µM,respectively. Furthermore, the bis-ANS displacement assay revealed that these compounds competitively inhibited the binding between the probe bis-ANS and the MD2 protein. The most active compound 3g, revealed a directly bind withMD2 proteinvia Arg90binding and a dissociation constant value of 1.52×10-6mol·L-1as determined by the biological layer interference (BLI) assay. Ourfinding suggestedthat compounds3gcould bea promising lead compound as MD2 inhibitor for further anti-inflammatory agent development.