Sodium-translocating NADH:quinone oxidoreductase (Na-NQR) functions as a unique redox-driven sodium pump, generating membrane potential, which is related to aminoglycoside antibiotic resistance. However, whether it modulates other metabolisms to confer antibiotic resistance is unknown. The present study showed that loss of or led to differential metabolomes with elevated resistance to aminoglycoside antibiotics. Decreased alanine, aspartate, and glutamate metabolism and depressed abundance of alanine were characterized as the most impacted pathway and crucial biomarker, respectively. Further data showed that higher viability was detected in Δ and Δ mutant strains than their parent strain ATCC 33787 in the presence of gentamicin but recovered by exogenous l-alanine. It proceeds by the following events. The loss of or led to the decrease of membrane potential, ATPase activity, and then ATP and cyclic AMP (cAMP), which reduced the cAMP/CRP (cAMP receptor protein) complex. The reduced cAMP/CRP complex promoted l-alanine catabolism and inhibited l-alanine anabolism, causing reduced levels of alanine. Reduced alanine affected the expression of antiporter families Atp and Mnh genes. Our results suggest a novel mechanism by which the Na-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner. The Na-NQR complex functions as a unique redox-driven sodium pump, generating membrane potential directly. However, whether it mediates generation of membrane potential indirectly is unknown. The present study shows that the Na-NQR complex impacts membrane potential through other antiporter families Atp and Mnh. It proceeds by ATP and then cAMP/CRP regulon, which inhibits l-alanine catabolism and promotes l-alanine anabolism. When the Na-NQR complex is reduced as in antibiotic-resistant bacteria, l-alanine is depressed, which is related to the antibiotic resistance phenotypes. However, exogenous l-alanine reverts the phenotype and promotes antibiotic-mediated killing. These findings suggest a novel mechanism by which the Na-NQR system regulates antibiotic resistance via l-alanine metabolism in a cAMP/CRP complex-dependent manner.