Hepatocellular carcinoma (HCC) is a common malignant tumor with poor prognosis. We previously showed that expression of Delta-like 3 (DLL3), a member of the family of Delta/Serrate/Lag2 ligands for the Notch receptor, is silenced by aberrant DNA methylation and that overexpression of DLL3 in an HCC cell line induces cellular apoptosis. However, how DLL3 expression is regulated during hepatocarcinogenesis is still unclear. Here, we show that silencing of DLL3 during hepatocarcinogenesis is closely related to viral infection, especially hepatitis B virus (HBV) infection (p = 0.005). HepG2.2.15 cells, which are stably transformed with the HBV genome, showed lower DLL3 expression than the parent cell line, HepG2 cells. Treatment with Hepatitis B virus X protein (HBx) small interfering RNA upregulated DLL3 expression in HepG2.2.15 cells, and overexpression of HBx in HepG2 cells downregulated DLL3 expression. Treatment of cells with a histone deacetylase inhibitor induced DLL3 expression in HepG2.2.15 cells. These data suggest that DLL3 expression is silenced during hepatocarcinogenesis in association with HBV infection via an epigenetic mechanism.