The recognition of cytoplasmic nucleic acid is critical for innate immune responses against microbial infection and is responsible for autoimmunity induced by dead cells. Here, we report the identification of a unique cytosolic nucleic acid cosensor in human airway epithelial cells and fibroblasts: DEAH (Asp-Glu-Ala-His) box polypeptide 29 (DHX29), a member of the DExD/H (Asp-Glu-x-Asp/His)-box helicase family. Knocking down DHX29 by siRNA attenuated the ability of cells to mount type I IFN and IL-6 in response to cytosolic nucleic acids and various viruses by blocking the activation of interferon regulatory factor 3 and NF-κB-p65. The cytosolic nucleic acid sensing by DHX29 in human epithelial cells and fibroblasts is independent of stimulator of interferon genes but is dependent on retinoic acid-inducible gene 1 (RIG-I) and mitochondrial antiviral signaling protein (MAVS). DHX29 binds directly to nucleic acids and interacts with RIG-I and MAVS through its helicase 1 domain, activating the RIG-I-MAVS-dependent cytosolic nucleic acid response. These results suggest that DHX29 is a cytosolic nucleic acid cosensor that triggers RIG-I/MAVS-dependent signaling pathways. This study will have important implications in drug and vaccine design for control of viral infections and viral-induced pathology in the airway.