Previous studies have reported that estrogen receptors (ERs) are expressed in normal human liver, chronic hepatitis, and benign hepatic tumor tissues. However, decreased expression of ERs can be observed in hepatocellular carcinoma (HCC) and the role of ERs in HCC is not fully understood. Thus, the present study aimed to investigate the molecular mechanism induced by the overexpression of ERa (ERa (ESR1)) in Hep3B cells. We first detected the induction of apoptosis in ER-negative Hep3B cells using DNA fragmentation assay and flow cytometry. We found that ERa and ERa plus 17b-estradiol treatment increased apoptosis in Hep3B cells. Additionally, western blotting showed increased expression of active caspase 3 and tumor necrosis factor a (TNFa (TNF)) in ERa-transfected cells. To further understand the importance of SP1-binding sites in the TNFa promoter, ERa-negative Hep3B cells were co-transfected with ERa and a wild-type TNFa plasmid or TNFa with deleted SP1 regions. Deletion of both distant and primal SP1 sites abolished the activity of ERa, and similar results were observed by blocking the expression of SP1 protein using mithramycin (MA). This result indicates that SP1 protein is essential for ERa-activated TNFa promoter activity. Co-immunoprecipitation assay further confirmed the binding interaction between ERa and SP1 in a ligand-dependent manner.In general, we demonstrate that the overexpression of ERa mediates apoptosis in ERa-negative Hep3B cells by the binding of ERa to SP1 protein. Additionally, this ERa-SP1 complex binds to the proximal and distal sites of the TNFa gene promoter and further induces the expression of active caspase 3 in a ligand-dependent manner.