Zinc is an essential nutritional factor involved in many key biological processes. However, the physiological function of zinc transporters at the organismal level is not well characterized. Early embryonic lethality of Znt1 knockout mice precludes functional analysis of the role of ZnT1 in dietary zinc absorption. Here, we report the identification and characterization of the Drosophila ZnT1 orthologue, dZnT1, for its role in Drosophila dietary zinc absorption. In cell culture, dZnT1 promoted zinc transport to reduce cytoplasmic zinc levels. Ubiquitous RNA interference of dZnT1 in Drosophila resulted in developmental arrest under restriction of dietary zinc, while dZnT1-overexpressing flies exhibited hypersensitivity to zinc. dZnT1 was prominently expressed in restricted regions of the midgut and exhibited a distribution on the basolateral membrane of the enterocytes. Gut-specific silencing of dZnT1 was sufficient to evoke lethality under zinc scarcity. Human ZnT1, but not ZnT7 or ZnT4, could rescue the zincacquiring defects caused by dZnT1 silencing. Taken together, our results proved that dZnT1 is a key zinc transporter in dietary zinc absorption, functioning by pumping zinc out of the enterocytes across the basolateral membrane. This study will be helpful in understanding the fundamental process of acquiring dietary zinc in higher eukaryotes.—Wang, X., Wu, Y., Zhou, B. Dietary zinc absorption is mediated by ZnT1 in Drosophila melanogaster. FASEB J. 23, 2650-2661 (2009)