Liver-derived hepcidin Hamp regulates iron homeostasis by downregulating ferroportin Fpn expression at the sites of iron absorption (duodenal enterocytes), recycling (reticuloendothelial macrophages) and storage (liver). However, Fpn and Hamp are also expressed in tissues not associated with systemic iron handling, such as the heart, skeletal muscle, lung and brain, raising the possibility of autocrine iron regulation in these tissues. Current mouse models of ubiquitous gene deletion do not enable us to address this hypothesis due to the confounding effects of ubiquitous loss of Hamp and Fpn on systemic iron levels. To overcome this limitation, we have used the LoxP Cre technology to enable tissue-specific manipulation of Hamp and Fpn expression in the mouse. Here we describe, for the first time, the generation and biochemical validation of three mouse models: 1) tissue-specific Hamp knockout, 2) tissue-specific Fpn knockout and 3) tissue-specific Fpn C326Y knock-in (hamp-resistant). Our animal models are valuable tools for micro-dissecting iron regulation at a single-tissue level in health and in disease. They will help achieve the mechanistic understanding necessary for the development of evidence-based iron manipulation therapies for diseases where iron dysregulation contributes to pathogenesis