Staphylococcus aureusis the most clinically relevant pathogen regarding implant-associated bone infection and its capability to invade osteoblasts is well known. The aim of this study was to investigate firstly whetherS. aureusis not only able to invade but also to proliferate within osteoblasts, secondly to delineate the mechanism of invasion and thirdly to clarify whether rifampicin or gentamicin can inhibit intracellular proliferation and survival ofS. aureus. The SAOS-2 osteoblast-like cell line and human primary osteoblasts were infected withS. aureusEDCC5055 andS. aureusRosenbach 1884. BothS. aureusstrains were able to invade efficiently and to proliferate within human osteoblasts. Immunofluorescence microscopy showed intracellular invasion ofS. aureusand transmission electron microscopy images could demonstrate bacterial division as a sign of intracellular proliferation as well as cytosolic bacterial persistence. Cytochalasin D, the major actin depolymerisation agent, was able to significantly reduceS. aureusinvasion, suggesting that invasion was enabled by promoting actin rearrangement at the cell surface. 7.5 μg/mL of rifampicin was able to inhibit bacterial survival in SAOS-2 cells with almost complete elimination of bacteria after 4 h. Gentamicin could also kill intracellularS. aureusin a dose-dependent manner, an effect that was significantly lower than that observed using rifampicin. In conclusion,S. aureusis not only able to invade but also to proliferate in osteoblasts. Invasion seems to be associated with actin rearrangement at the cell surface. Rifampicin is effective in intracellular eradication ofS. aureuswhereas gentamicin only poorly eliminates intracellularly replicating bacteria.