Hepatitis C virus (HCV) protease NS3/4A cleaves mitochondrial antiviral signaling protein (MAVS) to evade host innate recognition and interferon production. However, HCV infection induces interferon production in certain cell types and HCV could replicate in cells expressing NS3/4A cleavage-resistant MAVS. The roles of NS3/4A-mediated MAVS cleavage in host innate evasion and restriction of viral replication need to be clarified. In this study, by using an HCV NS3/4A-mediated MAVS cleavage reporter system, in which Huh7.5 cells express GFP-NLS-MAVS-TM462, we dissected the kinetics of MAVS cleavage during HCV infection. It was found that MAVS was barely cleaved during the early time but was efficiently cleaved only in the late time during viral infection. Using ypet-tagged HCV virus and Huh7.5 cells expressing RFP-NLS-MAVS-TM462, we dissected the MAVS cleavage in the HCV-positive (ypet-positive) cells on a single-cell manner. It was found that MAVS cleavage occurred only in part of the HCV-infected cells. We also assessed the MAVS cleavage by HCV NS3/4A from different genotypes, and found the cleavage efficiency was correlated with the protein level of HCV NS3/4A. Taken together, we found NS3/4A-mediated MAVS cleavage occurs in the late stage during viral replication and is correlated with NS3 protein level. We proposed HCV NS3/4A-mediated MAVS cleavage does not contribute to host innate evasion during early viral replication step.