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<i>Staphylococcus aureus </i>(<i>S. aureus</i>)<i> </i><i>nt5</i> gene, which encodes 5’-nucleotidase, is nearly unknown for its function in staphylococcal virulence and bacterial infection. In this study, we performed<i> nt5</i>^C166T gene mutation using a CRISPR RNA-guided base editing system, and further carried out transcriptome sequencing of the<i> </i>mutant strain to investigate the gene mutation response upon antibiotic daptomycin (DAP) exposure and its influence on bacterial virulence<i> in vitro</i> and <i>in vivo</i>. RNA sequencing showed that <i>nt5</i> inactivation inhibited bacterial nucleotide metabolism, and several nucleotide metabolism-related genes were significantly down-regulated in the mutant strain. The<i> </i>mutant strain enhanced tolerance to DAP treatment by attenuating cell membrane potential dissipation, slowing DNA release, and inhibiting nucleotide metabolism. Moreover, the <i>nt5</i> mutation alleviated the damage caused by bacterial infection in the kidney by reducing the expression of IL-β, IL-6 and IL-18 in mouse abscess models. Immune cells were more likely to swallow the mutant strain, which also led to lower bacterial toxicity of mutant<i> S. aureus </i>during the infection process. In summary, <i>nt5</i> mutation enhances the tolerance of <i>S. aureus</i> to antibiotics but impedes the bacterial infection capacity to form kidney abscesses by increasing<i> </i>bacterial phagocytosis by the host cell immune system <i>in vitro</i> and<i> in vivo</i>, which demonstrates <i>nt5</i> gene facilitates bacterial virulence and immune evasion in <i>S. aureus </i>pathogenesis.