Antiviral immunity against a pathogen is mounted upon recognition by the host of virally associated structures. One of these viral ‘signatures’, double-stranded (ds) RNA, is a replication product of most viruses within infected cells and is sensed by Toll-like receptor 3 (TLR3) and the recently identified cytosolic RNA helicases RIG-I (retinoic acid inducible gene I, also known as Ddx58) and Mda5 (melanoma differentiation-associated gene 5, also known as Ifih1 or Helicard 1.) Both helicases detect dsRNA, and through their protein-interacting CARD domains, relay an undefined signal resulting in the activation of the transcription factors interferon regulatory factor 3 (IRF3) and NF-κB. Here we describe Cardif, a new CARD-containing adaptor protein that interacts with RIG-I and recruits IKK-α, IKK-β and IKK-ϵ kinases by means of its C-terminal region, leading to the activation of NF-κB and IRF3. Overexpression of Cardif results in interferon-β and NF-κB promoter activation, and knockdown of Cardif by short interfering RNA inhibits RIG-I-dependent antiviral responses. Cardif is targeted and inactivated by NS3-4A, a serine protease from hepatitis C virus known to block interferon-β production. Cardif thus functions as an adaptor, linking the cytoplasmic dsRNA receptor RIG-I to the initiation of antiviral programmes.