Chitons (Polyplacophora) are intertidal molluscs that rasp algae from rocks using teeth coated with iron. Chitons produce these iron-clad teeth throughout their lifespans on a conveyor-belt like feeding organ called a radula. To better understand the molecular mechanisms chitons use to biomineralize iron, we sequenced the genome of the West Indian Fuzzy Chiton, Acanthopleura granulata. We found A. granulata has a greater number of genes potentially regulated by iron than other molluscs. We also investigated the physiological environment within the radula. We discovered an iron mineralization zone of 6-8 tooth rows in which A. granulata facilitates magnetite crystallization by lowering the pH of radula tissues to ~4. We expected chitons to use free iron (iron not bound to proteins) in radula biomineralization, but free iron causes oxidative stress. Consequently, we analyzed relative amounts of reactive oxygen species (ROS) across the radula of A. granulata and found more ROS in the iron-rich regions of the radula compared to regions without iron, indicating higher oxidative stress where iron biomineralization is taking place. Informed by physiology, we sequenced transcriptomes from developmentally distinct regions of the radula and characterized gene expression patterns associated with iron biomineralization. This is the first study of a chiton radula to use physiological information to guide analyses. Studies of chitons like A. granulata facilitate future investigations of the mechanisms and evolution of metal biomineralization in animals.