Familiar clustering of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) has been frequently reported. However, limited information is available about the underlying molecular mechanisms in HBV-related HCC patients with family history of HCC. In our previous study, Agilent miRNA Base 16.0 microarray showed miRNA profiles of the plasma of HBV-related HCC patients who had a family history of HCC. This study aims to explore the expression, function, and mechanisms of miR-3188 in HCC that might provide novel insights into the role of family history on the risk of HCC. The expression levels of miR-3188 were markedly overexpressed in HCC tissues, HBV transgenic mice, and HepG2.215 cells. We knocked out miR-3188 in HCC cell lines using the CRISPR/Cas9 system, and demonstrated that miR-3188 knockout (KO) suppressed cell growth, migration, and invasion, and inhibited xenografts tumor growth in nude mice. Next, we determined that miR-3188 KO exerts antitumor functions by directly repressing ZHX2. It has been reported that HBV X protein (HBx) plays a critical role in HBV-related HCC, promoting CREB-mediated activation of miR-3188 and activation of Notch signaling through repressing ZHX2. Finally, we verified that ZHX2 functions as a transcriptional repressor to Notch1 via interaction with NF-YA. Our data demonstrate that the HBx-miR-3188-ZHX2-Notch1 signaling pathway plays an important role in the pathogenesis and progression of HBV-related HCC with family history of HCC. These findings have important implications for identifying new therapeutic targets in HBV-related HCC.