Through shRNA library screening, we identified H-Ras as a positive regulator in antiviral responses, whose depletion caused increase of Sindbis virus replication, vesicular stomatitis virus infection and a decrease in Sendai virus (SeV)-induced RIG-I-like receptor (RLR) signaling. In experiments of ectopically expressing H-Ras, however, expression of wild-type H-Ras results in a biphasic regulation in RLR signaling: while low-level expression of H-Ras enhances SeV-induced RLR signaling, high-level expression of H-Ras significantly reduces RLR signaling. The inhibitory effects correlate with the activation status of H-Ras. As a result, oncogenic H-Ras, H-RasV12, strongly inhibits SeV-induced IFN-β promoter activity and type I interferon (IFN-I) signaling. Conversely, H-Ras exerted positive effect on RLR signaling which is independent of its signaling activity, as a constitutively inactive form of H-Ras, H-RasN17, also positively regulates RLR signaling. We also demonstrate that depletion of H-Ras reduces the formation of MAVS-TRAF3 signaling complexes. These results reveal that the H-Ras protein is participated in promoting MAVS signalosome formation, whereas oncogenic H-Ras exerted signaling negatively regulates type I IFN responses.