Copper, an essential trace element, becomes toxic when its concentration exceeds certain natural levels. Recent in vitro studies demonstrated that copper overload of cells may induce apoptosis through caspase-3 activation. However, the precise mechanism of copper-induced apoptosis in fish is still unclear, even less in O. niloticus where no studies have been reported so far. Accordingly, the main aim of this study was to find if copper induces apoptosis in Oreochromis niloticus gill, through the caspase pathway. Additionally, we intended to evaluate the potential use of activated caspase-3 expression as a waterborne copper biomarker.