The central role of the prion protein (PrP) in a family of fatal neurodegenerate diseases has garnered considerable research interest over the past two decades. Moreover, the role of PrP in neuronal development, as well as its apparent role in metal homeostasis, is increasingly of interest. The host-encoded form of the prion protein (PrPC) binds multiple copper atoms via its N-terminal domain and can influence brain copper and iron levels. The importance of PrPC to the regulation of brain metal homeostasis and metal distribution, however, is not fully understood. We therefore employed synchrotron-based X-ray fluorescence imaging to map the level and distributions of several key metals in the brains of mice that express different levels of PrPC. Brain sections from wild-type, prion gene knockout (Prnp−/−) and PrPC over-expressing mice revealed striking variation in the levels of iron, copper, and even zinc in specific brain regions as a function of PrPC expression. Our results indicate that one important function of PrPC may be to regulate the amount and distribution of specific metals within the central nervous system. This raises the possibility that PrPC levels, or its activity, might regulate the progression of diseases in which altered metal homeostasis is thought to play a pathogenic role such as Alzheimer's, Parkinson's and Wilson's diseases and disorders such as hemochromatosis.