Intracellular Staphylococcus aureus (S.aureus) often causes clinical failure and relapse after antibiotic treatment.We previously found that 20(S)-ginsenoside Rh2[20(S)-Rh2]enhanced the therapeutic effect of quinolones in a mouse model of peritonitis,which we attributed to the increased concentrations of quinolones within bacteria.In this study,we investigated the enhancing effect of 20(S)-Rh2 on levofloxacin (LVF) from a perspective of intracellular bacteria.In S.aureus 25923-infected mice,coadministration of LVF(1.5 mg/kg,i.v.) and 20(S)-Rh2 (25,50 mg/kg,i.g.) markedly increased the survival rate,and decreased intracellular bacteria counts accompanied by increased accumulation of LVF in peritoneal macrophages.In addition,20(S)-Rh2 (1,5,10 μaM) dose-dependently increased the uptake and accumulation of LVF in peritoneal macrophages from infected mice without drug treatment.In a model of S.aureus 25923-infected THP-1 macrophages,we showed that 20(S)-Rh2 (1,5,10 μM) dose-dependently enhanced the intracellular antibacterial activity of LVF.At the cellular level,20(S)-Rh2 increased the intracellular accumulation of LVF by inhibiting P-gp and BCRP.PK-PD modeling revealed that 20(S)-Rh2 altered the properties of the cell but not LVF.At the subcellular level,20(S)-Rh2 did not increase the distribution of LVF in lysosomes but exhibited a stronger sensitizing effect in acidic environments.Molecular dynamics (MD) simulations showed that 20(S)-Rh2 improved the stability of the DNA gyrase-LVF complex in lysosome-like acidic conditions.In conclusion,20(S)-Rh2 promotes the cellular pharmacokinetics and intracellular antibacterial activities of LVF against S.aureus through efflux transporter inhibition and subcellular stabilization,which is beneficial for infection treatment.