Primary and cryopreserved hepatocytes and immortalized hepatic cell lines in static two-dimensional monolayer culture format have been widely used as in vitro liver models for studies of xenobiotic metabolism, enzyme induction, hepatocyte regeneration, and hepatotoxicity. However, the tissue structure and metabolic capacity in these liver models are often ill-defined and are not well preserved compared to in vivo liver-specific architecture and functions. For this reason, we developed a three-dimensional (3D) dynamic flow model with primary human hepatocytes, which was optimized for cell seeding density, medium composition, and extracellular matrix proteins. Human hepatocytes cultured in this system were maintained for up to 7 weeks and reproducibly recapitulated in vivo liver-like structure and important liver-specific functions, such as albumin/total protein production, glucose utilization, lactate production, and cytochrome P450 (CYP) 3A4 activity across multiple tissue donors. The in vitro intrinsic clearance (CLint) of 7-ethoxycoumarin (7-EC) was determined from human hepatocytes cultured in the 3D dynamic flow model and compared to that in hepatocyte suspension. The 7-EC CLint values varied among individual batches and/or the two different in vitro liver models used in this study. The 3D flow model appeared to give more reproducible and stable estimates of clearance that is similar to previously published values. Overall, the results from these studies demonstrate that this culture system could be a valuable tool for making more accurate predictions of the metabolic clearance and long-term effects of chemicals and their metabolites in a complex 3D environment under dynamic flow.