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We developed a bioartificial liver (BAL) containing human hepatoblastoma cell line, HepG2, with the addition of ammonia removal activity by transfecting a glutamine synthetase (GS) gene and estimated the efficacy using pigs with ischemic liver failure. GS-HepG2 cells showed 15% ammonia removal activity of porcine hepatocytes, while unmodified HepG2 had no such activity. The established GS-HepG2 cells were grown in a circulatory flow bioreactor to 3.5–4.1 × 109 cells. Survival time of the animals treated with GS-HepG2 BAL was significantly prolonged compared to the cell-free control (14.52 ± 5.24 h vs. 8.53 ± 2.52 h) and the group treated with the BAL consisting of unmodified wild-type HepG2 (9.58 ± 4.52 h). Comparison showed the cell-containing BAL groups to have significantly fewer incidences of increased brain pressure. Thus, the GS-HepG2 BAL treatment resulted in a significant improvement of survival time and pathological parameters in pigs with ischemic liver failure.