The present study aimed to assess the possible preventive and reparative effects of isoleucine (Ile) against copper (Cu)-induced oxidative stress in fish enterocytes in vitro. In experiment 1, enterocytes were preincubated with increasing concentrations of Ile (0, 50, 120, 190, 260, and 330 mg L-1) for 72 h followed by exposure to 6 mg L-1 Cu for 24 h. In experiment 2, the enterocytes were pretreated with 6 mg L-1 Cu for 24 h and then treated with 0-330 mg L-1 Ile for 72 h to investigate its potential reparative role. The results of experiment 1 showed that Cu exposure increased lactate dehydrogenase (LDH) activity and malondialdehyde and protein carbonyl (PC) content; these changes were completely suppressed by pretreatment with Ile at optimum concentrations (P < 0.05). Moreover, Ile pretreatment prevented the decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities in the enterocytes exposed to Cu (P < 0.05). Additionally, cells exposed to Cu exhibited adaptive increases in glutathione-S-transferase (GST) activity. In experiment 2, the LDH activity and protein oxidation induced by Cu were completely reversed by Ile posttreatment. Meanwhile, the Cu-induced decrease in SOD, GPx, and GST activity was completely reversed by subsequent Ile treatment, but the reduced glutathione content was not restored. Collectively, these results indicate that Ile suppresses Cu-induced oxidative damage via preventive and reparative pathways in primary enterocytes and thus protects the structural integrity of enterocytes in fish.