An immortalized human hepatocyte cell line, HepLi5, was established via transfection of Simian virus 40 large T antigen (SV40 LT) into primary human hepatocytes. The morphologic and functional characteristics of HepLi5 cells were evaluated. The expression of SV40 LT in HepLi5 cells was detected by reverse transcription-PCR (RT-PCR) and western blotting. mRNA expression of liver-enriched genes, including glutamine synthetase, albumin, and cytochrome P450 was detected via RT-PCR in HepLi5 cells. Activity of CYP1A2, one of the drug-metabolizing P450 enzymes, was detected. Subcutaneous injection of HepLi5 cells into nude mice did not induce tumors within 3 months. Short Tandem Repeat results confirmed the authenticity of the cell line. Clinical-grade quantities of HepLi5 cells could be harvested using large-scale culture in roller bottles after which their cellular function was significantly enhanced. Therefore, the immortalized HepLi5 cells are a suitable cell source for applications in bioartificial livers.