To engineer liver tissues with a clinically significant size, in vivo evaluation of performance using large-scale animal studies are necessary before proceeding to human clinical trials. As pigs are the most suitable candidates, the development of culture conditions suitable for porcine hepatocyte progenitors is very important to engineer pig liver tissue equivalents. We therefore investigated the efficacy of poly-L-lactic acid (PLLA) three-dimensional (3D) scaffolds on the functional maturation of fetal porcine hepatocytes in the presence of various combinations of biofactors. Cells were isolated from pig fetuses obtained from a local slaughterhouse, and cultured for 15 days both in monolayer and PLLA scaffolds. Although 15 days of culture resulted in almost the same ratio of proliferation (about fivefold) in both monolayer and 3D PLLA culture, the PLLA culture with hepatocyte growth factor (HGF, 10 ng/ml) and sodium butylate (Sb, 1 mM) remarkably enhanced various liver-specific functions of fetal porcine hepatocytes. The final attained functions based on the numbers of immobilized cells on day 1 compared with those of day 1 monolayers; 191-fold increase in albumin secretion, 70.5-fold increase in cytochrome P450 IA1/2 capacity, 20.9-fold increase in ammonia removal, and 18.0-fold increase in urea synthesis were obtained. These functions were 2.0-3.3-fold higher than those obtained by the same period of monolayer culture. In addition, final attained unit cell-based functions on day 15 were almost comparable to the levels reported for cultures of adult porcine hepatocytes in both monolayer and 3D spheroid cultures. These results demonstrate that the use of a biodegradable polymer-based 3D culture with an appropriate combination of biofactors is a promising approach to maximize functional maturation of hepatocyte progenitors from large animals. In addition, the established culture conditions are worth using to engineer large liver tissue equivalents for pigs in large-animal-based preclinical studies.