ATP-sensitive potassium channels (K ATP channels) are critical nutrient sensors in many mammalian tissues. In the pancreas, K ATP channels are essential for coupling glucose metabolism to insulin secretion. While orthologous genes for many components of metabolism–secretion coupling in mammals are present in lower vertebrates, their expression, functionality and ultimate impact on body glucose homeostasis are unclear. In this paper, we demonstrate that zebrafish islet β-cells express functional K ATP channels of similar subunit composition, structure and metabolic sensitivity to their mammalian counterparts. We further show that pharmacological activation of native zebrafish K ATP using diazoxide, a specific K ATP channel opener, is sufficient to disturb glucose tolerance in adult zebrafish. That β-cell K ATP channel expression and function are conserved between zebrafish and mammals illustrates the evolutionary conservation of islet metabolic sensing from fish to humans, and lends relevance to the use of zebrafish to model islet glucose sensing and diseases of membrane excitability such as neonatal diabetes.