Trace minerals such as copper (Cu) and zinc (Zn) are animal nutrition supplements necessary for livestock health and breeding performance, yet they also have environmental impacts via animal excretion. Here we investigated changes in Cu and Zn speciation from the feed additive to the broiler excreta stages. The aim of this study was to assess whether different Cu and Zn feed additives induce different Cu and Zn speciation patterns, and to determine the extent to which this speciation is preserved throughout the feed-animal-excreta system. Synchrotron-based X-ray absorption spectroscopy (XAS) was used for this investigation. The principal findings were: (i) in feed, Cu and Zn speciation changed rapidly from the feed additive signature (Cu and Zn oxides or Cu and Zn sulfates) to Cu and Zn organic complexes (Cu phytate and Zn phytate). (ii) in the digestive tract, we showed that Cu and Zn phytate were major Cu and Zn species; Cu sulfide and Zn amorphous phosphate species were detected but remained minor species. (iii) in fresh excreta, Cu sulfide and Zn amorphous phosphate were major species. These results should help to: (i) enhance the design of future research studies comparing different feed additive performances; (ii) assess Cu and Zn bioavailability in the digestive tract; (iii) gain further insight into the fate of Cu and Zn in cultivated soils when poultry manure is used as fertilizer.