Exhaustion of cytotoxic effector natural killer (NK) and CD8+ T cells have important functions in the establishment of persistent viral infections, but how exhaustion is induced during chronic hepatitis C virus (HCV) infection remains poorly defined. Here we show, using the humanized C/OTg mice permissive for persistent HCV infection, that NK and CD8+ T cells become sequentially exhausted shortly after their transient hepatic infiltration and activation in acute HCV infection. HCV infection upregulates Qa-1 expression in hepatocytes, which ligates NKG2A to induce NK cell exhaustion. Antibodies targeting NKG2A or Qa-1 prevents NK exhaustion and promotes NK-dependent HCV clearance. Moreover, reactivated NK cells provide sufficient IFN-γ that helps rejuvenate polyclonal HCV CD8+ T cell response and clearance of HCV. Our data thus show that NKG2A serves as a critical checkpoint for HCV-induced NK exhaustion, and that NKG2A blockade sequentially boosts interdependent NK and CD8+ T cell functions to prevent persistent HCV infection.