Primary hepatocytes have been used in drug development for the evaluation of hepatotoxicity of candidate compounds. However, the rapid depression of their hepatic characters in vitro must be improved to predict toxicity with higher accuracy. We have hypothesized that a well organized tissue construct that includes nonparenchymal cells and appropriate scaffold material(s) could overcome this difficulty by remediating the viability and physiological function of primary hepatocytes. In this study, we constructed an in vitro liver tissue model, consisting of mouse primary hepatocytes assembling around an endothelial cell network on Engelbreth-Holm-Swarm gel, and examined its response to acetaminophen treatment. The increase in lactate dehydrogenase release after the exposure to acetaminophen was induced earlier in the liver tissue model than in monolayer hepatocytes alone, suggesting that the tissue model was more sensitive to an acetaminophen-induced toxicity. On the basis of our results, we conclude that liver tissue models of this kind may enhance the responses of hepatocytes against xenobiotics via the maintenance of hepatic genes and functions such as cytochrome P450s. These findings will contribute to the development of more accurate systems for evaluating hepatotoxicity.