Anthropogenic activities in agriculture and health use the antimicrobial properties of copper. This has led to copper accumulation in the environment and contributed to the emergence of copper resistant microorganisms. Understanding bacterial copper homeostasis diversity is therefore highly relevant since it could provide valuable targets for novel antimicrobial treatments. The periplasmic CopI protein is a monodomain cupredoxin comprising several copper binding sites and is directly involved in copper resistance in bacteria. However, its structure and mechanism of action are yet to be determined. To study the different binding sites for cupric and cuprous ions and to understand their possible interactions, we have used mutants of the putative copper binding modules of CopI and spectroscopic methods to characterize their properties. We show that CopI is able to bind a cuprous ion in its central histidine/methionine-rich region and oxidize it thanks to its cupredoxin center. The resulting cupric ion can bind to a third site at the N-terminus of the protein. Nuclear magnetic resonance spectroscopy revealed that the central histidine/methionine-rich region exhibits a dynamic behavior and interacts with the cupredoxin binding region. CopI is therefore likely to participate in copper resistance by detoxifying the cuprous ions from the periplasm.