Hepatitis C Virus NS3/NS4A, a serine protease complex, has been found to interact with many host proteins and cause various adverse effects on cellular function and immune response. For example, the cleavage of important immune factors by NS3/NS4A has been suggested as a mechanism for the hepatitis C virus to evade innate immunity. The spectrum of susceptible substrates for NS3/NS4A cleavage certainly includes important immune modulator kinases such as IKKα, IKKβ, IKKε, and TBK1, as demonstrated in this paper. We show that the kinase activities of these four host kinases were transformed in unexpected ways by NS3/NS4A. Treatment with NS3/NS4A caused a significant reduction in the kinase activities of both IKKα and IKKβ, suggesting that HCV might use its NS3/NS4A protease activity to deactivate the NF-κB-associated innate immune responses. In contrast, the kinase activities of both IKKε and TBK1 were enhanced after NS3/NS4A treatment, and more strikingly, the enhancement was more than 10-fold within 20 min of treatment. Our mass spectroscopic results suggested that the cleavage after Cys89 in the kinase domain of IKKε by NS3/NS4A led to their higher kinase activities, and three potential mechanisms were discussed. The observed kinase activity enhancement might facilitate the activation of both IKKε- and TBK1-dependent cellular antiviral pathways, likely contributing to spontaneous clearance of the virus and observed acute HCV infection. After longer than 20 min cleavage, both IKKε- and TBK1 gradually lost their kinase activities and the relevant antiviral pathways were expected to be inactivated, facilitating the establishment of chronic HCV infection.