Retinoic acid-inducible gene I (RIG-I) is responsible for innate immunity via the recognition of short double-stranded RNAs in the cytosol. With the clue that G-U wobble base pairs in the influenza A virus's RNA promoter region are responsible for RIG-I activation, we determined the complex structure of RIG-I Delta CARD and a short hairpin RNA with G-U wobble base pairs by X-ray crystallography. Interestingly, the overall helical backbone trace was not affected by the presence of the wobble base pairs; however, the base pair inclination and helical axis angle changed upon RIG-I binding. NMR spectroscopy revealed that RIG-I binding renders the flexible base pair of the influenza A virus's RNA promoter region between the two G-U wobble base pairs even more flexible. Binding to RNA with wobble base pairs resulted in a more flexible RIG-I complex. This flexible complex formation correlates with the entropy-favoured binding of RIG-I and RNA, which results in tighter binding affinity and RIG-I activation. This study suggests that the structure and dynamics of RIG-I are tailored to the binding of specific RNA sequences with different flexibility.