The liver plays a central role in all metabolic processes in our body. Currently, orthotropic liver transplantation is the only proven effective treatment for serious acute liver failure (ALF) such as fulminant hepatic failure (FHF). However, liver transplantation has not yet become a standard treatment for FHF because of a shortage of donors. A bioartificial liver (BAL) device containing living hepatocytes might be a therapeutic alternative to liver transplantation. In this study, we developed a hollow fiber (HF)-type bioartificial liver (BAL) module and evaluated the performance of an embryonic stem cell (ESC)-immobilized bioartificial liver (ES-BAL) module in vitro and ex vivo. Mouse ESCs were immobilized in a BAL module and then cultured under perfusion conditions. Cells immobilized inside the HFs had a high proliferative activity and formed cylindrical organoids. For hepatic differentiation of cultured cells, differentiation-promoting agents were added to the culture medium. Ammonia removal and albumin secretion were detected after about 2 weeks of culture. Male Wistar rats were used in animal experiments. After induction of liver failure, rats were connected to an ES-BAL module containing differentiated ESCs or a control module (without cells) and underwent extracorporeal circulation for 1 h. We measured the changes in blood biochemical parameters. All rats in the control group died within 10 h, whereas two out of three rats treated with the ES-BAL module recovered after the operation. We evaluated the changes in blood biochemical parameters and the liver weight of surviving rats treated with the ES-BAL module. Most of the blood biochemical parameters had returned to normal ranges at 1 week after treatment. We also observed increased liver weight. In conclusion, we developed a novel HF-type BAL module containing differentiated ESCs with liver-specific functions. Our BAL module has the potential to support liver functions and induce liver regeneration in rats with liver failure.