Abstract : Insulin-secreting pancreatic beta cells are exceptionally rich in zinc. In these cells, zinc is required for zinc-insulin crystallization within secretory vesicles. Secreted zinc has also been proposed to be a paracrine and autocrine modulator of glucagon and insulin secretion in pancreatic alpha and beta cells, respectively. However, little is known about the molecular mechanisms underlying zinc accumulation in insulin-containing vesicles. We previously identified a pancreas-specific zinc transporter, ZnT-8, which colocalized with insulin in cultured beta cells. In this paper we studied its localization in human pancreatic islet cells, and its effect on cellular zinc content and insulin secretion. In human pancreatic islet cells, ZnT-8 was exclusively expressed in insulin-producing beta cells, and colocalized with insulin in these cells. ZnT-8 overexpression stimulated zinc accumulation and increased total intracellular zinc in insulin-secreting INS-1E cells. Furthermore, ZnT-8-overexpressing cells display enhanced glucose-stimulated insulin secretion compared with control cells, only for a high glucose challenge, i.e. >10 mM glucose. Altogether, these data strongly suggest that the zinc transporter ZnT-8 is a key protein for both zinc accumulation and regulation of insulin secretion in pancreatic beta cells.