Abstract Background A great many patients awaiting liver transplantation die because of the inavailability of donor livers. Liver support systems such as the bioartificial liver have been effective alternatives to ease the shortage. However, the problem with these systems is the difficulty to obtain sufficient amounts of hepatocytes with good liver function. This study explored the possibility of employing a human fetal hepatocyte cell line L-02 for a liver support system. Objective To confirm the hepatocytic functions of L-02 cells and their potential as a novel cell source to treat acute liver failure (ALF) in a liver support system. Methods Hepatocyte markers were detected by Western blotting and laser confocal microscopy; their gene expression of hepatic markers was examined by polymerase chain reaction (PCR). An in vivo ALF model was established by 90% partial hepatectomy. Ten rats undergoing hepatectomy received 5 × 107 L-02 cells intrasplenically and 15 served as controls. Survival and liver functions were assessed in both groups. Results In vitro, Western blotting and laser confocal microscopy showed L-02 to express liver function markers: Albumin (ALB), uridine diphosphate glucuronosyltransferase, and cytochrome P450 3A4. PCR showed the expression of liver-specific genes, such as ALB, human glutathione S transferase, and human factor-X. In vivo, rats transplanted with L-02 cells showed significantly improved survival of 70% versus 0% in controls (P < .01). Liver function in the L-02 group was significantly improved versus controls: ALB, 30.2143 ± 2.68665 versus 25.3 ± 7.27942 g/L; alanine transaminase, 1611.333 ± 342.0078 versus 2831.333 ± 110.437 U/L; aspartate aminotransferase, 2210.667 ± 97.57732 versus 3149.333 ± 71.98842 U/L; serum ammonia, 360.4667 ± 74.94656 versus 660.62 ± 63.41681 μmol/L; alkaline phosphates, 408.6667 ± 48.00347 versus 698.5 ± 17.67769 U/L; total bilirubin, 29.8 ± 6.19785 versus 44.6 ± 9.73858 μmol/L; and direct bilirubin, 25.4333 ± 6.60631 versus 32.5 ± 7.07107 μmol/L (P < .05). Conclusion L-02 cells, expressing the main molecules of human hepatocytes, improved liver functions and survival of ALF rats, suggesting them to be a feasible source for liver support systems.