Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Long cytosolic dsRNA is recognized by MDA5. The cooperative assembly of MDA5 into helical filaments on dsRNA nucleates the assembly of a multiprotein type I interferon signaling platform. Here, we determined cryoelectron microscopy (cryo-EM) structures of MDA5-dsRNA filaments with different helical twists and bound nucleotide analogs at resolutions sufficient to build and refine atomic models. The structures identify the filament-forming interfaces, which encode the dsRNA binding cooperativity and length specificity of MDA5. The predominantly hydrophobic interface contacts confer flexibility, reflected in the variable helical twist within filaments. Mutation of filament-forming residues can result in loss or gain of signaling activity. Each MDA5 molecule spans 14 or 15 RNA base pairs, depending on the twist. Variations in twist also correlate with variations in the occupancy and type of nucleotide in the active site, providing insights on how ATP hydrolysis contributes to MDA5-dsRNA recognition. Display omitted •Cryo-EM structures of MDA5-dsRNA filaments determined for three catalytic states•Filament forming interfaces are flexible and predominantly hydrophobic•Mutation of filament-forming residues can cause loss or gain of IFN-β signaling•ATPase cycle is coupled to changes in filament twist and size of the RNA footprint Cryo-EM structures determined by Yu et al. show how the innate immune sensor MDA5 forms helical filaments to recognize cytosolic double-stranded RNA. Mutations at the hydrophobic filament interface can cause loss or gain of signaling activity. ATP binding and hydrolysis cause structural changes, which are proposed to confer proofreading activity.