Lys63-linked polyubiquitination of RIG-I is essential in antiviral immune defense, yet the molecular mechanism that negatively regulates this critical step is poorly understood. Here, we report that USP21 acts as a novel negative regulator in antiviral responses through its ability to bind to and deubiquitinate RIG-I. Overexpression of USP21 inhibited RNA virus-induced RIG-I polyubiquitination and RIG-I-mediated interferon (IFN) signaling, whereas deletion of USP21 resulted in elevated RIG-I polyubiquitination, IRF3 phosphorylation, IFN-α/β production, and antiviral responses in MEFs in response to RNA virus infection. USP21 also restricted antiviral responses in peritoneal macrophages (PMs) and bone marrow-derived dendritic cells (BMDCs). USP21-deficient mice spontaneously developed splenomegaly and were more resistant to VSV infection with elevated production of IFNs. Chimeric mice with USP21-deficient hematopoietic cells developed virus-induced splenomegaly and were more resistant to VSV infection. Functional comparison of three deubiquitinases (USP21, A20, and CYLD) demonstrated that USP21 acts as a bona fide RIG-I deubiquitinase to down-regulate antiviral response independent of the A20 ubiquitin-editing complex. Our studies identify a previously unrecognized role for USP21 in the negative regulation of antiviral response through deubiquitinating RIG-I.