Enterovirus D68 (EV-D68) is a member of the Picornavirus family and a causative agent of respiratory diseases in children. The incidence of EV-D68 infection has increased worldwide in recent years. Thus far, there are no approved antiviral agents or vaccines for EV-D68. Here, we show that methyl-β-cyclodextrin (MβCD), a common drug that disrupts lipid rafts, specifically inhibits EV-D68 infection without producing significant cytotoxicity at virucidal concentrations. The addition of exogenous cholesterol attenuated the anti-EV-D68 activity of MβCD. MβCD treatment had a weak influence on the attachment of viral particles to the cell membrane but significantly inhibited EV-D68 entry into host cells. We demonstrated that EV-D68 facilitated the translocation of the viral receptor ICAM-5 to membrane rafts in infected cells. The colocalization of viral particles with ICAM-5 in lipid rafts was thoroughly abolished in cells after treatment with MβCD. Finally, we showed that MβCD inhibited the replication of isolated circulating EV-D68 strains. In summary, our results demonstrate that MβCD suppresses EV-D68 replication by perturbing the accumulation of virus particles and ICAM-5 in lipid rafts. This mechanism represents a promising strategy for drug development.,Copyright © 2020 Elsevier B.V. All rights reserved.