The pathogen is the causative agent of cholera. Emergence of antibiotic-resistant strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in We found that N16961, a pandemic strain, and its isogenic (p)ppGpp-overexpressing mutant ΔΔ are both more antibiotic-resistant than (p)ppGpp (ΔΔΔ) and Δ mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B-encoding and tricarboxylic acid (TCA) cycle gene in the (p)ppGpp mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including , was increased in (p)ppGpp, but not in the antibiotic-resistant ΔΔ mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate-binding protein FbpA was increased 10-fold in (p)ppGpp, and gene deletion restored viability of tetracycline-exposed (p)ppGpp cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in .