Novel adjuvants are needed to increase the efficacy of vaccine formulations and immune therapies for cancer and chronic infections. In particular, adjuvants that promote a strong type I IFN response are required, since this cytokine is crucial for the development of efficient anti-tumoral and anti-viral immunity. Nucleic acid band 2 (NAB2) is a double-stranded RNA molecule isolated from yeast and identified as an agonist of the pattern-recognition receptors TLR3 and MDA-5. We compared the ability of NAB2 to activate innate immunity with that of poly(I:C), a well-characterized TLR3 and MDA-5 agonist known for the induction of type I IFN. NAB2 promoted stronger IFN-α production and induced a higher activation state of both murine and human innate immune cells compared to poly(I:C). This correlated with a stronger activation of the signalling pathway downstream of MDA-5, and IFN-α induction was dependent on MDA-5. Upon injection, NAB2 induced higher levels of serum IFN-α in mice than poly(I:C). These results suggest that NAB2 has the potential to become an efficient adjuvant for the induction of type-I IFN responses in therapeutic immunization against cancer or infections.