Hepatitis B virus (HBV) infection is a major public health issue with serious medical consequences. The roles of microRNAs (miRNAs) in HBV replication and expression have been generally recognized, and the abnormal expression of miR-501 has been reported in patients with HBV infection. However, the function and mechanism in HBV replication remain elusive. The expression patterns of miR-501-3p and ZEB2 in HBV-related hepatocellular carcinoma (HCC) tissues and HBV-expressing HCC cells were determined by qRT-PCR and western blot assays. HBV replication and expression were evaluated through detecting the copies of HBV DNA and the secretion of HBV surface antigens HBsAg and HBeAg by real-time PCR and ELISA analyses. Bioinformatics and dual-luciferase reporter analyses were employed to validate the functional interaction between miR-501-3p and ZEB2. miR-501-3p was significantly upregulated, while ZEB2 was downregulated in HBV-related HCC tissues and cells compared with relative controls without HBV infection. Knockdown of miR-501-3p hampered HBV replication and gene expression in HepG2.2.15 and HepAD38 cells. ZEB2 was identified as a functional target of miR-501-3p. The absence of ZEB2 abolished the inhibitory effects of anti-miR-501-3p on HBV replication and gene expression. Our data indicated that miR-501-3p participated in the regulation of HBV replication and gene expression partially via repressing ZEB2 in HepG2.2.15 and HepAD38 cells, providing a promising antiviral avenue for HBV infection. © 2020, AEPress, s.r.o. All rights reserved.