Hepatitis C virus (HCV) often establishes a persistent infection that most likely involves a complex host-virus interplay. We previously reported that the HCV nonstructural protein 5A (NS5A) bound to cellular protein FKBP38 and resulted in apoptosis suppression in human hepatoma cell line Huh7. In the present research we further found that NS5A increased phosphorylation levels of two mTOR-targeted substrates, S6K1 and 4EBP1, in Huh7 in the absence of serum. mTOR inhibitor rapamycin or NS5A knockdown blocked S6K1 and 4EBP1 phosphorylation increase in NS5A-Huh7 and HCV replicon cells, suggesting that NS5A specifically regulated mTOR activation. Overexpression of NS5A and FKBP38 mutants or FKBP38 knockdown revealed this mTOR activation was dependent on NS5A-FKBP38 interaction. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 treatment in NS5A-Huh7 showed that the mTOR activation was independent of PI3K. Moreover, NS5A suppressed caspase 3 and poly(ADP-ribose) polymerase activation, which was abolished by NS5A knockdown or rapamycin, indicating NS5A inhibited apoptosis specifically through the mTOR pathway. Further analyses suggested that apoptotic inhibition exerted by NS5A via mTOR also required NS5A-FKBP38 interaction. Glutathione S-transferase pulldown and co-immunoprecipitation showed that NS5A disrupted the mTOR-FKBP38 association. Additionally, NS5A or FKBP38 mutants recovered the mTOR-FKBP38 interaction; this indicated that the impairment of mTOR-FKBP38 association was dependent on NS5A-FKBP38 binding. Collectively, our data demonstrate that HCV NS5A activates the mTOR pathway to inhibit apoptosis through impairing the interaction between mTOR and FKBP38, which may represent a pivotal mechanism for HCV persistence and pathogenesis.