Background and Aims Acetaminophen (APAP) is a safe and effective antipyretic, analgesic, and anti-inflammatory drug. However, excessive use of APAP can rapidly induce acute liver injury and liver failure, leading to patient death or the need for liver transplantation. Studies have shown that macrophages are essential in maintaining liver homeostasis and regulating the progression of acute and chronic liver injuries. Therefore, this study investigated the role and mechanisms of macrophage functional changes in APAP-induced acute liver injury.Methods Male BALB/c mice were randomly divided into the control group (gavage with normal saline), drug-induced liver injury model group (APAP group, 600 mg/kg APAP gavage), and drug-induced liver injury model plus macrophage depletion agent clodronate liposomes (CL) group (APAP+CL group, intravenous injection of CL 12 h before APAP gavage). After 3 h of APAP gavage, serum and liver tissue samples were collected from each group of mice. The serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured. Liver tissue pathological changes were observed, and the levels of reactive oxygen species (ROS) and heme oxygenase-1 (HO-1) expression in the liver tissue were detected. RAW246.7 cells were cultured with APAP or APAP plus HO-1 inhibitor zinc protoporphyrin Ⅸ (ZnPPⅨ) using untreated RAW246.7 cells as a control. Then, the changes in cellular ROS levels and HO-1 expression were observed.Results The results of the animal experiment showed that compared to the control group, the APAP group exhibited significant pathological changes in the liver, with significant increases in serum levels of ALP, ALT, and AST, as well as elevated levels of ROS and HO-1 expression in liver tissue. However, in the APAP+CL group, these changes were significantly suppressed, and the differences in all quantitative indicators were statistically significant (all P<0.05). The cell experiment results showed that ROS levels and HO-1 expression in RAW246.7 cells were significantly increased after incubation with APAP. However, when co-incubated with ZnPPⅨ, the APAP-induced ROS levels and HO-1 expression elevation were significantly inhibited (all P<0.05).Conclusion APAP may promote macrophage ROS generation by inducing HO-1 expression, causing acute liver injury. Intervention targeting this pathway may provide a new approach to preventing and treating clinical acute liver injury.