Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture.