Infections such as biomaterial-associated infection and osteomyelitis are often associated with intracellular survival of bacteria (eg, Staphylococcus aureus). Treatment of these infections remains a major challenge due to the low intracellular efficacy of many antibiotics. Therefore, local delivery systems are urgently required to improve the therapeutic efficacy of antibiotics by enabling their intracellular delivery. To assess the potential of gelatin nanospheres as carriers for local delivery of vancomycin into macrophages of zebrafish larvae in vivo and into THP-1-derived macrophages in vitro using fluorescence microscopy. Fluorescently labeled gelatin nanospheres were prepared and injected into transgenic zebrafish larvae with fluorescent macrophages. Both the biodistribution of gelatin nanospheres in zebrafish larvae and the co-localization of vancomycin-loaded gelatin nanospheres with zebrafish macrophages in vivo and uptake by THP-1-derived macrophages in vitro were studied. In addition, the effect of treatment with vancomycin-loaded gelatin nanospheres on survival of S. aureus-infected zebrafish larvae was investigated. Internalization of vancomycin-loaded gelatin nanospheres by macrophages was observed qualitatively both in vivo and in vitro. Systemically delivered vancomycin, on the other hand, was hardly internalized by macrophages without the use of gelatin nanospheres. Treatment with a single dose of vancomycin-loaded gelatin nanospheres delayed the mortality of S. aureus-infected zebrafish larvae, indicating the improved therapeutic efficacy of vancomycin against (intracellular) S. aureus infection in vivo. The present study demonstrates that gelatin nanospheres can be used to facilitate local and intracellular delivery of vancomycin.