RIG-I plays important roles in pathogen sensing and activation of antiviral innate immune responses in response to RNA viruses. RIG-I-mediated signaling must be precisely controlled to maintain innate immune signaling homeostasis. Previous studies demonstrated that lysine 63 (K63)-linked polyubiquitination of RIG-I is vital for its activation, but the mechanisms through which RIG-I is deubiquitinated to control innate immune responses are not well understood. Here we identified USP27X as a negative regulator of antiviral signaling in response to RNA viruses through siRNA library screening. Further functional studies indicated that USP27X negatively modulated RIG-I-mediated antiviral signaling in a deubiquitinase-dependent manner. Mechanistically, we found that USP27X removed K63-linked polyubiquitin chains from RIG-I to negatively modulate type I interferon signaling. Collectively, these studies uncover a novel negative regulatory role of USP27X in targeting RIG-I to balance innate immune responses.