Acetaminophen (APAP) overdose can lead to acute kidney injury (AKI), yet its molecular mechanisms remain unclear and no effective treatments are currently available. In this study, we combined transcriptomic, proteomic, and phosphoproteomic profiling of kidneys from APAP-exposed mice to explore molecular mechanisms and potential therapeutic strategies. Ten-week-old male C57BL/6 mice were fasted overnight for 16 hours prior to APAP treatment. Acute kidney injury was induced by intraperitoneal injection of APAP overdose (300 mg/kg body weight) for 6 hours (n = 4). Control mice received an equivalent volume of PBS via intraperitoneal injection (n = 4). Kidney tissues were subsequently collected from APAP-induced kidney injury mice and PBS-injected controls.