Isorhamnetin is a hepatoprotective flavonoid molecule derived from the leaves and fruits of Hippophae rhamnoides L. However, the protective effect of isorhamnetin on acetaminophen (APAP) induced hepatotoxicity is still unknown. Thus, we aimed to investigate the lipid-lowering, anti-inflammatory, and hepatoprotective effects of isorhamnetin on APAP-induced hepatotoxicity in mice. Hepatotoxicity was induced by a single injection of APAP (300 mg/kg, intraperitoneally). Isorhamnetin (50 or 100 mg/kg, orally) and N-acetylcysteine (NAC) (200 mg/kg, orally), or vehicle control, were administered 1 h before the administration of APAP. Total antioxidant status (TAS) and total oxidative status (TOS) of liver tissue and levels of inflammatory factors (TNF-α, IL-1β, and IL-6) were analyzed by ELISA. Lipid profiles and liver function parameters were measured using an autoanalyzer. In addition, liver tissue was examined histopathologically. Isorhamnetin treatment significantly reduced the APAP-induced increase in the liver weight and liver index; it also reduced the APAP-induced increase in serum liver parameters (ALT, AST, ALP, and LDH) (p < 0.05). Isorhamnetin significantly reduced APAP-induced oxidative stress and inflammation by increasing TAS levels and decreasing TOS, TNF-α, IL-1β, and IL-6 levels (p < 0.05). Moreover, isorhamnetin treatment significantly regulated lipid profiles (TG, T-C, LDL-C, and HDL-C levels) that changed in response to APAP administration (p < 0.05). In histopathological examination, liver degeneration observed in the APAP group was significantly reduced in the NAC and isorhamnetin-treated groups (p < 0.05). This study suggests that isorhamnetin has a significant protective effect on APAP-induced hepatotoxicity in mice through its lipid-lowering, antioxidant, and anti-inflammatory effects.