Hepatitis B virus X protein (HBx) is highly expressed in HBV-infected hepatocellular carcinoma (HCC) and upregulates transcriptional activation of telomerase reverse transcriptase (TERT). NHP2 is a component of the telomerase complex and also increased in HCC. However, whether NHP2 could accelerate HCC caused by HBx overexpression remains unknown. This study intended to investigate the effects of NHP2 knockdown on HBx-overexpressed HCC and uncover the potential mechanism. Results showed that after HBx overexpression, the expression of TERT and NHP2 was increased. NHP2 knockdown inhibited cell proliferation, colony formation and telomerase activity, while promoting cell apoptosis in PLC/PRF5 cells with or without HBx overexpression. Moreover, the protein expression of TERT and HBx was inhibited, pro-apoptotic proteins Bax and cleaved-caspase3 expression was enhanced, whereas anti-apoptotic protein Bcl-2 level was reduced upon NHP2 silencing in PLC/PRF5 cells with or without HBx upregulation. The interaction between NHP2 and TERT was also confirmed. Treatment with shRNA-NHP2-1 inhibited tumor growth in xenograft model, and the alterations of related proteins were consisted with in vitro results. In conclusion, knockdown of NHP2 could inhibit the proliferation of hepatoma cells overexpressing HBx via inhibiting TERT expression.