It is well known that a three-dimensional (3D) culture environment and the presence of extracellular matrix (ECM) proteins facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro. However, it is not clear whether specific ECM components such as collagen or fibronectin differentially regulate such processes, especially in 3D scaffolds. In this study, a series of ECM-functionalized inverted colloidal crystal (ICC) microporous scaffolds were fabricated and their influence on Huh-7.5 cell proliferation, morphology, hepatic-specific functions, and patterns of gene expression were compared. Both collagen and fibronectin promoted albumin production and liver-specific gene expression of Huh-7.5 cells, compared with the bare ICC scaffold. Interestingly, cells in the fibronectin-functionalized scaffold exhibited different aggregation patterns to those in the collagen-functionalized scaffold, a variation that could be related to the distinct mRNA expression levels of cell adhesion-related genes. Based on these results, we can conclude that different ECM proteins, such as fibronectin and collagen, indeed play distinct roles in the phenotypic regulation of cells cultured in a 3D environment.