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Eprinomectin (EPM), a member of avermectin family, is a semi-synthetic antibiotic. It has been known that avermectin family enters the aquatic environments and adversely affects the aquatic organisms. Effects of EPM is fully unknown in aquatic organisms especially fish, thus the aim of the present study was to investigate transcriptional changes (sod, cat, gpx) and activities of some antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) and malondialdehyde (MDA) levels, oxidative DNA damage (8-hydroxy-2-deoxyguanosine (8-OHdG)) and transcriptional changes of heat shock protein 70 (HSP70), and cytochromes P4501A (CYP1A) in liver tissues of rainbow trout exposed to sublethal EPM concentration (0.001 μg/L, 0.002 μg/L, 0.01 μg/L, 0.05 μg/L) for 24 h, 48 h, 72 h and 96 h.The decrease in antioxidant enzyme (SOD, CAT and GPx) activity, transcriptional changes (sod, cat, gpx, HSP70 and CYP1A genes) and increase in MDA level and activity of 8-OHdG in a dose–time-dependent manner in the liver of rainbow trout were observed. The down-regulated of antioxidant (sod, cat and gpx), HSP70 and CYP1A obviously, the severity of which increased with the concentration of EPM and exposure time. The results imply that EPM could induce oxidative damage to the liver tissue of rainbow trout. The information presented in this study is helpful to understand the mechanism of veterinary pharmaceuticals–induced oxidative stress in fishes.EPM has biochemical and physiological effects in fish liver.These parameters may be involved in the toxicity mechanism of EPM on rainbow trout.8-OHdG, CYP1A, HSP70 and antioxidant parameters might be utilized as a potential indicator of exposure to antibiotic in fish.