Intracellular bacteria often lead to chronic and recurrent infections; however, most of the known antibiotics have poor efficacy against intracellular bacteria due to their poor cell membrane penetration efficiency into the cytosol. Here, a thiol-mediated nanodrug delivery system, named Van-DM NPs, was developed to improve vancomycin's penetration efficiency and intracellular antibacterial activities. Van-DM NPs were prepared through self-assembly of vancomycin (Van) and the disulfide molecule (DM) in NaOH buffer solution. On the one hand, the disulfide exchange reaction between Van-DM NPs and the bacterial surface enhances vancomycin accumulation in bacteria, increasing the local concentration of vancomycin. On the other hand, the disulfide exchange reaction between Van-DM NPs and the mammalian cell membrane triggered the translocation of Van-DM NPs across the mammalian cell membrane into the cell cytosol. These dual mechanisms promote antibacterial activities of vancomycin against both extracellular and intracellular bacteria S. aureus. Furthermore, in an intravenous S. aureus infection mouse model, Van-DM NPs exhibited high antibacterial capability and efficiently reduced the bacterial load in liver and spleen, where intracellular bacteria tend to reside. Altogether, the reported Van-DM NPs would be highly promising against intracellular pathogenic infections.