Pycnogenol® (PYC) has already being used as a food supplement and herbal medicine due to its potent antioxidant properties. The aim of the present study was to examine the protective effect of PYC on acetaminophen‐induced acute liver injury in rats. The effect of PYC on acetaminophen‐induced hepatotoxicity in rats was examined by determining biochemical parameters, in vitro antioxidant activity, histological assessment, and oxidative status in liver homogenates. The best antioxidant properties were demonstrated in methanolic extracts. Seven‐day pretreatment with PYC suppressed elevation of CYP2E1 protein expression induced by administration of toxic dose of acetaminophen. PYC at 50 mg/kg showed the ability to significantly decrease malondialdehyde (MDA) level compared with the group received acetaminophen. Xanthine oxidase (XOD) enzyme activity was significantly elevated in acetaminophen‐treated group compared with control, whereas concomitant administration of PYC in a dose of 50 mg/kg significantly reduced activity of this enzyme. Significant decrease of glutathione (GSH) hepatic content in acetaminophen‐intoxicated rats compared with the control rats was improved by concomitant administration of PYC at 50 mg/kg. Protective effect of PYC on acetaminophen‐induced acute liver injury in rats has showed the best in vitro antioxidant potential expressed in methanolic extract and consequent histological assessment and oxidative status in liver homogenates.