Although oxidative stress has been demonstrated to be involved in copper (Cu)-induced toxicity, information regarding the effect of antioxidants on Cu toxicity is still scarce. This study assessed the possible protective effects of myo-inositol (MI) against subsequent Cu exposure in juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and in their enterocytes in vitro. First, oxidative stress was established by exposing fish to different concentrations of Cu (0-7.2 mg Cu/L water) for 4 days. Next, the protective effects of MI (administered as a dietary supplement for 60 days) against subsequent Cu exposure (0.6 mg Cu/L water for 4 days) were studied in fish. The third trial determined the effects of Cu exposure (0-6.0 mg Cu/L of medium for 24h) on enterocytes in vitro. Finally, enterocytes were pre-incubated with graded levels of MI (0-75 mg MI/L of medium) for 72 h and exposed to 6.0 mg Cu/L of medium for 24h. The results indicated that ≥ 0.6 mg Cu/L water could induce oxidative stress in fish (P<0.05). Cu exposure significantly induced increases in lipid peroxidation and protein oxidation in the gill, hepatopancreas and intestine in fish. However, these oxidative effects were prevented by MI pre-supplementation. MI also prevented the toxic effects of Cu on anti-superoxide anion (ASA), anti-hydroxyl radical (AHR), superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in these organs. In vitro, enterocytes exposed to Cu displayed a dose-dependent injury. Moreover, cell viability, protein retention (PR), alkaline phosphatase, total-SOD (T-SOD) and Cu/ZnSOD activities were all depressed by Cu (P<0.05). Interestingly, the final experiment showed that MI pre-supplementation could block the toxic effects of Cu on the antioxidant system, and thus protect enterocytes from Cu-induced oxidative damage. All of these results indicated that the induction of key antioxidant defenses by MI pre-supplementation, including SOD, CAT, GPx, GST and GSH, may play an important role in the protection of fish against oxidative stress.