Objective:The aim of this study was to investigate the underlying mechanism whereby HBx modulates the targeting ofNUSAP1 bymiR-18b to enhance hepatocarcinogenesis. Methods:We employed an integrated approach of bioinformatics analysis and molecular experiments in hepatoma cells, HBV transgenic mice, and clinical liver cancer tissues to investigate the role of HBx-regulatedmiR-18b in the development of liver cancer. Results:In this study, we report that the HBx-mediated tumor suppressormiR-18b modulates hepatocarcinogenesis during the host-HBV interaction. The expression levels ofmiR-18b were lower in clinical HBV-positive liver cancer tissues and liver tissues of HBV-transgenic mice. Interestingly, HBx inhibitedmiR-18b expression by inducing the methylation of CpG islands in its promoter. Accordingly, we tested the hypothesis that HBx enhanced hepatocarcinogenesis by increasing the expression of target genes ofmiR-18b. Moreover, we identified nucleolar spindle-associated protein 1 (NUSAP1) as one of the target genes ofmiR-18b. NUSAP1 was expressed at high levels in liver cancer tissues. Interestingly, HBx up-regulated NUSAP1 by suppressingmiR-18b. Functionally,miR-18b significantly inhibited the proliferation of hepatoma cells by depressing NUSAP1 levelsin vivo andin vitro. Conclusions:Thus, we conclude that the targeting ofNUSAP1 mRNA by the tumor suppressormiR-18b is controlled by HBx-modulated promoter methylation during the host-virus interaction, leading to hepatocarcinogenesis. Our findings provide new insights into the mechanism by which HBx-mediated miRNAs modulate hepatocarcinogenesis.