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Glutamate metabolism plays a pivotal role in linking the tricarboxylic acid cycle, arginine biosynthesis, and purine metabolism and these pathways have been shown to be involved in persister formation. However, the relationship among glutamate metabolism, bacterial antibiotic tolerance, and virulence remains unclear. In this study, <i>gltB</i> which encodes the large subunit of glutamate synthase in <i>Staphylococcus aureus </i>was knocked out. The ∆<i>gltB</i> mutant in the stationary phase showed less tolerance to antibiotics and was killed completely after exposure to lethal doses of ampicillin and norfloxacin after 11 and 6 days, respectively, while the parent strain still had abundant viable bacteria. The <i>gltB</i> complemented strain restored antibiotic tolerance. Interestingly, exogenous glutamate supplementation of ∆<i>gltB</i> restored the tolerance to antibiotics.<b><i> </i></b>Moreover, ∆<i>gltB</i> is more susceptible to heat, carbon source deficiency, and oxidative stress. In addition, ∆<i>gltB</i> attenuated virulence in BALB/c mice, and its LD50 (1.1 × 10¹⁰CFU/mL) was 4.6 times higher than that of the parent strain (2.3 × 10⁹ CFU/mL). Furthermore, the ability of ∆<i>gltB</i> to coagulate plasma, produce staphyloxanthin, and form biofilms was significantly weakened. The expression levels of major virulence genes, including<i> eta, hla, hlgA, hlgC, lukD, lukE, lukS, lukF, </i>and <i>sea,</i> as well as staphyloxanthin synthesis-related genes, including<i> </i><i>crt</i><i>M</i> and c<i>r</i><i>tQ</i> were significantly downregulated in ∆<i>gltB</i>. This study revealed that <i>gltB</i> is involved in both antibiotic tolerance and virulence in <i>S. aureus</i> and provides new insights into the mechanism of persister formation and virulence, with implications for the development of new drugs.