The aim of this study was to characterise biomarker responses in three-spined sticklebacks exposed to copper. For this purpose, adult sticklebacks were exposed for 3 weeks to copper sulphate at 0, 25, 100 and 200μgL(-1) as Cu. At days 4, 8, 12 and 21, several parameters were measured including liver, gonad and spleen somatic indexes, hepatic biomarkers (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), glutathione-S-transferase (GST) and 7-ethoxyresorufin-O-deethylase (EROD)) and hepatic copper and zinc concentrations. Copper induced a rapid and transient increase of antioxidant enzymes and a depletion of glutathione content during the first 8 days of exposure. Significant copper and zinc accumulation in fish liver were observed for the two higher exposure concentrations after 8 and 12 days, respectively. This study showed that copper induced an oxidative stress in fish liver before significant metal accumulation in the liver could be detected, suggesting the involvement of differential mechanisms in copper uptake and metabolism. Three-spined stickleback appears to be a sensitive model to study oxidative stress induced by metals.