Metals like iron (Fe) and copper (Cu) function as integral components in many biological reactions and in excess, these essential metals are toxic, and organisms must control metal acquisition and excretion. We examined the effects of chronic waterborne Cu exposure and the interactive effects of elevated dietary Fe on gene expression and tissue metal accumulation in zebrafish. Softwater acclimated zebrafish exposed to 8µg/L Cu, with and without supplementation of a diet high in Fe (560 vs 140 mg Fe/kg food) for 21d demonstrated a significant reduction in liver and gut Cu load relative to waterborne Cu exposure alone. Gene expression levels for divalent metal transport (DMT)-1, copper transporter (CTR)-1 and the basolateral metal transporter ATP7A in the gills and gut increased when compared to controls, but the various combinations of Cu and high Fe diet revealed altered levels of expression. Further examination of the basolateral Fe transporter, ferroportin, showed responses to waterborne Cu exposure in the gut, and a significant increase with Fe treatment alone in the liver. Additionally, we examined metallothionein 1 & 2 (MT1 & MT2), which indicated that MT2 is more responsive to Cu load. To explore the relationship between transcription and protein function, we examined both CTR-1 protein levels and gill apical uptake of radiolabelled Cu⁶⁴, which demonstrated decreased Cu uptake and protein abundance in the elevated Cu treatments. This study shows that high dietary Fe can significantly alter the genetic expression pattern of Cu transporters at the level of the gill, liver, and gastrointestinal tract.