Host cells orchestrate the production of IFN-β upon detecting invading viral pathogens. Here, we report that Ring finger protein 166 (RNF166) potentiates RNA virus-triggered IFN-β production. Overexpression of RNF166 rather than its homologous proteins RNF114, RNF125 and RNF138, enhanced Sendai virus (SeV)-induced activation of the IFN-β promoter. Knockdown of endogenous RNF166, but not other RNFs, inhibited the IFN-β production induced by SeV and encephalomyocarditis virus. RNF166 interacted with TRAF3 and TRAF6. SeV-induced ubiquitination of TRAF3 and TRAF6 was suppressed when endogenous RNF166 rather than RNF114/138 was knocked down. These findings suggest that RNF166 positively regulates RNA virus-triggered IFN-β production by enhancing the ubiquitination of TRAF3 and TRAF6.