Context: Camellia nitidissima Chi (Theaceae) is an evergreen shrub, the leaves of which are used in many medicinal applications. Objective: To characterize the chemical composition of a 10% aqueous ethanol extract of C. nitidissima leaves (CNE), and to explore the protective effect of the extract against acute liver injury (ALI) in rats. Materials and methods: Male Sprague-Dawley rats were divided into six groups (n ¼ 10): control and negative (0.5% CMC-Na, 5 mL/kg/d), thiopronin (20 mg/kg/d) and CNE (40, 80 and 160 mg/kg/d). All groups were treated for seven consecutive days, and then, except for the control, carbon tetrachloride was administered intraperitoneally. The biochemical parameters, mRNAs, and proteins were analyzed using enzyme-linked immunoassays kits, quantitative polymerase chain reaction and western blot. Chemical components were identified using mass spectroscopy, and the phenol and flavonoid content determined by ultraviolet spectrophotometry. Results: Pre-treatment with CNE (160 mg/kg) attenuated the pathological changes in liver tissues and decreased alanine transaminase (62 and 60%), aspartate transaminase (49 and 53%) and malondialdehyde (35 and 42%) levels in serum and liver tissues. Moreover, CNE reduced the concentrations of reactive oxygen species (55%), tumour necrosis factor-a (26%), interleukin-1b (19%) and IL-6 (19%) and blocked the nuclear translocation of p65. Pre-treatment with CNE increased anti-heme oxygenase-1 (40%), superoxide dismutase (108%) and glutathione (97%) levels through upregulating nuclear factor erythroid-2-related factor 2. Twelve compounds were detected; the content of phenols and flavonoids was determined as 34.474 ± 1.026 and 15.228 ± 0.422 mg/g crude drug in CNE, respectively. Discussion and conclusions: These results suggested that CNE is a promising agent for functional food and hepatoprotective drug against ALI.