Despite the identification of many positive and negative regulators that control RIG-I function [36, 37], the knowledge on the functional regulation of MDA5 is scanty. In order to identify proteins involved in MDA5 regulation, we performed a functional screening of a cDNA expression sub-library for their ability to inhibit or enhance type I IFN signaling [38–42]. For this purpose, human embryonic kidney (HEK) 293T cells (293T cells) were cotransfected with the IFN-β promoter luciferase reporter, Renilla luciferase internal control (pRL-TK), and cDNA expression plasmids followed by stimulation with intracellular HMW Poly(I:C), low molecular weight (LMW) Poly(I:C), or intracellular DNA duplex Poly(dA:dT). Among them, DHX29 was identified as a positive regulator of intracellular HMW Poly(I:C)-induced IFN-β-Luc reporter activity as well as interferon-stimulated response element (ISRE)-luciferase (Luc) reporter activity, which only requires interferon regulatory factor 3 (IRF3) activation (Fig 1A). In contrast, DHX29 failed to increase ISRE-Luc and IFN-β-Luc activities in TLR3-expressing 293T cells (293T-TLR3) stimulated with LMW Poly(I:C) or 293T cells stimulated with Poly(dA:dT) or intracellular LMW Poly(I:C) (Fig 1B–1D). Consistent with these results, the ectopic expression of DHX29 markedly increased IFN-β transcription as well as TBK1 and IRF3 phosphorylation in response to intracellular HMW Poly(I:C), but not to intracellular LMW Poly(I:C) or Poly(dA:dT) treatment (Fig 1E and 1F). Similar increase in IFIT1 (encodes ISG56), IFIT2 (encodes ISG54), and CCL5 mRNA expression, was observed in response to intracellular HMW Poly(I:C) in DHX29 overexpressed cells (S1A–S1C Fig). Taken together, these results strongly suggest that DHX29 specifically enhances MDA5, but not TLR3 or RIG-I, mediated type I IFN signaling pathway.