Emerging evidence has shown that hepatitis B virus (HBV) X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma. Complement regulatory proteins including CD46, CD55 and CD59 contribute to escape of tumor cells from complement-dependent cytotoxicity (CDC). However, little is known about the potential role of HBx in anti-CDC activity during hepatocarcinogenesis. In the present study, we for the first time report that HBx decreases the sensitivity of hepatoma and hepatic cells to CDC. Coincidentally, we demonstrated that HBx increased the promoter activity of CD59, as well as their messenger RNA and protein levels. Moreover, flow cytometry showed the increased expression level of CD59 protein on the surface of HBx-positive cells. Of interest, we found that HBx up-regulated CD59 by binding with cAMP response element-binding to the promoter region of the CD59 gene using chromatin immunoprecipitation assay. In addition, we showed that HBx up-regulated CD59 by let-7i at post-transcriptional regulation level. Our data showed that the deposition of C5b-9 were decreased on the cell surface in HepG2-X cells relative to HepG2 cells, suggesting that increased CD59 mediated by HBx prevents the formation of functional membrane attack complex. Furthermore, we demonstrated that down-regulation of CD59 was sufficient to abolish the resistance capability of CDC in HBx-positive cells by RNA interference (siRNA) in vitro and in vivo. Thus, we conclude that HBx contributes to cells resistance to CDC through CD59. Therapeutically, CD59 may serve as a target in HBV-associated hepatoma patients.