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.　A　thiol-mediated　nanodrug　delivery　system　was　developed　to　improve　the　cell　membrane　penetration　efficiency　and　intracellular　antibacterial　activities　of　antibiotics.