ABSTRACTCopper is an essential micronutrient but is toxic at high concentrations. InHaemophilus influenzaemechanisms of copper resistance and its role in pathogenesis are unknown; however, our previous genetic screen by transposon insertion-site sequencing implicated a putative cation transporting ATPase (copA) in survival in a mouse lung infection model. Here, we demonstrate thatH. influenzaecopA(HI0290) is responsible for copper homeostasis involving themerR-type regulator,cueR, as well as six tandem copies of the metallochaperone gene,copZ. Deletion of the ATPase and metallochaperone genes resulted in increased sensitivity to copper but not to cobalt, zinc, or manganese. NontypeableH. influenzae(NTHi) clinical isolate NT127 has the same locus organization but with three copies ofcopZ. We showed that expression of the NTHicopZAoperon is activated by copper under the regulatory control of CueR. NTHi singlecopAandcopZmutants and, especially, the double deletioncopZAmutant exhibited decreased copper tolerance, and the ΔcopZAmutant accumulated 97% more copper than the wild type when grown in the presence of 0.5 mM copper sulfate. Mutants of NT127 deleted of the ATPase (copA) alone and deleted of both the ATPase and chaperones (copZ1-3) were 4-fold and 20-fold underrepresented compared to the parent strain during mixed-infection lung challenge, respectively. Complementation ofcoplocus deletion mutations restored copper resistance and virulence properties. NTHi likely encounters copper as a host defense mechanism during lung infection, and our results indicate that thecopsystem encodes an important countermeasure to alleviate copper toxicity.