Copper is an essential element for normal cellular processes in most eukaryotic organisms, but is toxic in excessive amounts. Different organisms vary in their ability to tolerate copper ions. We have previously studied the mechanism of copper toxicity to a copper tolerance cell line, Hepa T1, from tilapia using a proteomic approach. To compare the differences of proteins' regulation between copper tolerant and sensitive species after copper treatment, the zebrafish liver cell line (ZFL) was used as a model in this study to investigate the mechanism of copper toxicity to zebrafish. After conducting similar experimental procedures in previous Hepa T1 studies, 72 different proteins were identified to be regulated by Cu(2+) (100 μM and 200 μM). More than 50% of these proteins were also found with differentially expressed Hepa T1, indicating that the toxicity mechanism between zebrafish and tilapia was partially conserved. However, the regulation of several proteins in ZFL, related to the reactive oxygen species (ROS) effect, mitochondrion copper transportation and stress response, was quite different from that in tilapia.