The aim of this study was to investigate the hepatoprotective effect of ultrafine powder of Dendrobium officinale (UDO) against acetaminophen (APAP)-induced liver injury in mice. ICR mice were orally administered with UDO (100, 200, 400 mg/kg/day) for 14 days before intraperitoneal injection of APAP. The results showed that UDO exhibited a significant dose-dependently preventive effect on APAP-induced hepatotoxicity. Pretreatment with UDO significantly reduced the serum levels of alanine transaminase and aspartate aminotransferase. Histological examination indicated that administration with UDO alleviated inflammation and necrosis as well as ameliorated hepatocyte steatosis, especially at 200 and 400 mg/kg doses. Additionally, UDO markedly recovered the activities of glutathione, catalase and total antioxidant capacity, and decreased the content of malonaldehyde and reactive oxygen species. In summary, the results suggested that pretreatment with UDO exists potently hepatoprotective effects against APAP-induced liver damage, which may be related to the enhanced antioxidant capacity and inhibited lipid peroxidation.