AIMS: The ability to eliminate bacterial persister cells is still a medical challenge that has yet to be overcome. These cells represent a unique subpopulation within bacterial communities and are characterized by a reduced susceptibility to antibiotics with growth retardation. In this study, we investigated the molecular basis of persister formation in Salmonella Typhimurium 14028 s under aminoglycoside stress. METHODS: We analyzed the crystal structure of the STM14_5441-STM14_5442 complex, which belongs to the type II toxin-antitoxin system, and identified key ribosome-binding residues in STM14_5441. Changes in the antibiotic susceptibility of Salmonella caused by the loss of the ribosome-binding property of STM14_5441 were assessed. We conducted intracellular ATP assays under aminoglycoside stress and RNA-seq analysis following STM14_5441 induction. RESULTS: Our studies demonstrated the critical role of STM14_5441 in the formation of persister cells in Salmonella, particularly those under aminoglycoside stress. We observed that a loss of ribosome binding in STM14_5441 resulted in increased antibiotic susceptibility. Additionally, intracellular ATP assays revealed increased ATP levels in STM14_5441 induced group, and RNA-seq analysis identified several genes that play a role in this phenomenon. CONCLUSIONS: The present data suggest that persister forms under aminoglycoside stress through the following mechanisms: i) inhibition of membrane hyperpolarization by impeding F