Autophagy has been shown to facilitate replication of hepatitis C virus (HCV); however, the mechanism by which HCV induces autophagy has not been fully established. NS5A, a nonstructural protein expressed by HCV, regulates numerous cellular pathways, including autophagy, by up-regulating Beclin 1; however, the underlying mechanism remains unclear. To obtain new insights into HCV-regulated autophagy, NS5ATP9 was overexpressed in HepG2 and L02 cells, resulting in up-regulation of endogenous Beclin 1 mRNA and protein levels, respectively. The luciferase-reporter assay results showed that both NS5A and NS5ATP9 could transactivate Beclin 1 promoter activity, but that NS5A could not transactivate the Beclin 1 promoter in NS5ATP9-silenced HepG2 and L02 cells. Up-regulation of Beclin 1 mRNA and protein expression by NS5A could also be attenuated by NS5ATP9 knock-down. Furthermore, the HepG2 and L02 cells that transiently overexpressed NS5ATP9 had enhanced accumulation of vacuoles carrying the autophagy marker LC3, consistent with the conversion of endogenous LC3-I to LC3-II. In contrast, the conversion of endogenous LC3-I to LC3-II could not be enhanced by NS5A in NS5ATP9-silenced HepG2 cells. These results highlight an important potential role for NS5ATP9 in HCV NS5A-induced hepatocyte autophagy.