The induction of type I interferon (IFN) is critical for antiviral innate immune response. During virus infection, Retinoic acid-inducible gene I (RIG-I) recognizes the viral RNA and activates Mitochondrial antiviral-signaling protein (MAVS). MAVS further triggers downstream signaling to activate Interferon regulatory factor 3 (IRF3) to induce type I IFN. Many viral proteases cleave adapter proteins in the IFN induction pathway, such as HCV NS3/4A cleaving MAVS, as a strategy to evade anti-viral innate immunity. However, it is not clear whether host protease may also regulate IFN induction pathway to control the steadily low expression of IFN in uninfected cells. Here we report that Hepsin (HPN), which belongs to type II transmembrane serine protease family, could inhibit the production of type I IFN during RNA virus infections in several cell lines. Hepsin expression could be detected in Huh7 and HepG2 hepatocytes but not ubiquitously in all cell types. Knocking-out Hepsin in mouse embryonic fibroblasts resulted in higher expression of IFNß and interferon response genes (ISGs). Besides, when co-transfected with N-RIG, which is constitutively activating IFNß expression, Hepsin could decrease IFNß promoter activity. However, under the expression of IRF3-5D, Hepsin could not reduce IFNß promoter activity, suggesting that Hepsin might target certain adaptor proteins downstream of RIG-I and upstream of IRF3. According to the substrate preference, we considered that Hepsin might cleave Stimulator of Interferon Genes (STING). Indeed, we showed an inverse correlation between STING and wild-type Hepsin co-expression but not the protease-deficient mutant Hepsin. When an in vitro cleavage assay was performed, we found that this phenotype could only be reproduced when membrane component was supplemented in the reaction. Consequently, these results reveal a novel role of Hepsin in regulating the type I IFN induction pathway in hepatocytes.