The casein kinase 1 (CK1) plays an important role in various biological processes by phosphorylating its target proteins. In this study, we demonstrate that CK1γ1 inhibits RNA virus–mediated activation of retinoic acid–inducible gene I (RIG-I) signaling by affecting the stability of NF-κB subunit p65. First, we found that ectopic expression of CK1γ1 inhibits RIG-I pathway–mediated activation of IFN-β, whereas knockdown of CK1γ1 potentiates the activation of IFN-β and NF-κB induced by Sendai virus (SeV). We then revealed that CK1γ1 interacts with p65 and specifically enhances its phosphorylation at Ser 536 induced by SeV. By using an in vitro kinase assay, we confirmed that CK1γ1 can phosphorylate p65 at Ser 536. We also showed that the kinase dead mutants CK1γ1K73A and CK1γ1N169A did not inhibit SeV-induced activation of IFN-β and NF-κB, suggesting that the kinase activity of CK1γ1 is critical for its inhibitory effect on RIG-I signaling. Additionally, we found that CK1γ1 also has the similar effect on TLR signaling. Further analysis indicated that CK1γ1 phosphorylates p65 and consequently promotes its degradation by ubiquitin E3 ligases CUL2 and COMMD1. These results revealed a novel negative regulatory manner of CK1γ1 on innate immune signaling.