Hovenia dulcis Thunb. is a traditional hepatoprotective Chinese medicine, and in research, much effort has been focused on the protection against alcoholic liver injury. In this study, the protective effects of a fruit ethanol extract of Hovenia dulcis (FE) against APAP-induced acute hepatotoxicity in mice and the possibly involved molecular mechanisms were investigated. Hepatoprotective activity of FE is clearly indicated by histopathological and biochemical examination. Treatment with FE resulted in inhibition of CYP2E1 activity involved in the transformation of APAP in vivo. Expressions of the altered bile acid metabolism and transport-related genes and relative proteins of apoptosis were normalized by preconditioning with FE before APAP treatment. These results suggested FE to alleviate APAP-induced liver injury in a dose-dependent manner by inhibition of cytochrome P450 activity, hepatocyte apoptosis and regulation of bile acid homeostasis imbalance.