<p>Pathogens are exposed to toxic levels of copper during infection, and copper tolerance may represent a general virulence mechanism utilized by bacteria to evade host defenses. Therefore, the precise regulation of copper homeostasis in bacteria is crucial for coping with external copper stress. However, little is known about the role of copper homeostasis in the virulence of <i>Klebsiella pneumoniae</i> (<i>K. pneumoniae</i>), an important clinical pathogen. This study focused on the potential systems of copper homeostasis in <i>K. pneumoniae</i> and found that deletion of Cue system genes significantly reduced bacterial copper tolerance. The decreased tolerance was due to increased intracellular copper accumulation and greater susceptibility to oxidative stress in the mutants. Furthermore, Δ<i>copA</i>, Δ<i>cueO</i> Δ<i>copA</i>, and Δ<i>cueO</i> Δ<i>cusRS</i> mutants may also be associated with reduced virulence and increased susceptibility to phagocytosis or killing by macrophages. Notably, the decreased virulence of Δ<i>copA</i> and Δ<i>cueO</i> Δ<i>copA</i> mutants may be associated with impaired capsule polysaccharide and biofilm formation. The Δ<i>cueO</i> Δ<i>cusRS</i> mutant also appeared to display increased sensitivity to hydrogen peroxide. Importantly, these attenuated mutants demonstrated protective effects and may serve as potential vaccine candidates. Altogether, this study identifies the critical role of copper homeostasis in bacterial copper tolerance and the virulence of <i>K. pneumoniae</i>.</p>