A series of 2-Benzoxyl-Phenylpyridine derivatives were evaluated for their potential antiviral activities against EV71. The preliminary assays indicated that some of these compounds exhibited excellent antiviral effects on EV71, they could effectively inhibit virus-induced cytopathic effects (CPEs), reduce progeny viral yields, and present similar or better antiviral activities compared to the positive control drug ribavirin. Among these derivatives, compounds WY7, WY13 and WY14 showed the most potency against EV71. Investigation of the underlying mechanism of action revealed that these compounds target EV71 replication in cells post infection, they could profoundly inhibit viral RNA replication and protein synthesis, and inhibit virus-induced cell apoptosis. Further experiments demonstrated that compound WY7 potently inhibited the activity of the EV71 3C protease (3Cpro), and to some extent, it affected the activity of 3D polymerase (3Dpol), thus blocking viral replication, but not the activity of the 2A proteinase (2Apro). Modeling of the molecular binding of the 3Cpro-WY7 complex revealed that compound WY7 was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity. These results indicate that these compounds might be feasible therapeutic agents against EV71 infection and that these compounds may provide promising lead scaffolds for the further design and synthesis of potential antiviral agents.Copyright © 2023. Published by Elsevier B.V.