Abstract: Acetaminophen (APAP, also known as paracetamol)-induced liver injury is the leading cause of drug-induced liver injury in the world. Wuzhi Tablet (WZ, an ethanol extract of Schisandra sphenanthera) is widely used in clinical practice to protect liver function. Our previous studies have shown that pretreatment with WZ for 3 days can significantly protect against APAP-induced liver injury; however, the effect of different intervals between APAP and WZ treatment on APAP-induced liver injury remains unclear. In this study, the change in liver injury indexes, APAP metabolites, and the activity of cytochrome P450 (CYP450) enzymes after treatment with WZ and APAP at different intervals were determined. The animal experiment was reviewed and approved by the Animal Ethics Committee of Sun Yat-sen University. The results show that 0 h, 0.5 h, and 2 h pretreatment with WZ significantly protected against APAP-induced liver injury in mice, as evidenced by a significant decrease in biochemical parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and malonaldehyde (MDA). WZ inhibited the metabolic activation of APAP mediated by CYP450 enzymes and reduced the formation of APAP metabolites. This study further demonstrates that pretreatment with WZ at different intervals (0 h, 0.5 h, and 2 h before APAP dosing) exerts a significant hepatoprotective effect against APAP-induced liver injury, and a single-dose of WZ inhibits the activity of CYP450 enzymes related to APAP metabolic activation, thereby protecting against APAP-induced hepatotoxicity.