Filoviruses often cause terrible infectious disease which has not been successfully dealt with pharmacologically. All filoviruses encode a unique protein termed VP35 which can mask doubled-stranded RNA to deactivate interferon. The interface of VP35–dsRNA would be a feasible target for structure-based antiviral agent design. To explore the essence of VP35–dsRNA interaction, molecular dynamics simulation combined with MM-GBSA calculations were performed on Marburg virus VP35–dsRNA complex and several mutational complexes. The energetic analysis indicates that nonpolar interactions provide the main driving force for the binding process. Although the intermolecular electrostatic interactions play important roles in VP35–dsRNA interaction, the whole polar interactions are unfavorable for binding which result in a low binding affinity. Compared with wild type VP35, the studied mutants F228A, R271A, and K298A have obviously reduced binding free energies with dsRNA reflecting in the reduction of polar or nonpolar interactions. The results also indicate that the loss of binding affinity for one dsRNA strand would abolish the total binding affinity. Three important residues Arg271, Arg294, and Lys298 which makes the largest contribution for binding in VP35 lose their binding affinity significantly in mutants. The uncovering of VP35–dsRNA recognition mechanism will provide some insights for development of antiviral drug. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 849–860, 2014.