Apis mellifera Royal Jelly (RJ) is a well-known remedy in traditional medicine around the world and its versatile effects range from antibacterial to anti-inflammatory properties and pro-regenerative properties. Several active compounds have been identified, however, the mechanisms of action still remain widely unknown. As a glandular product, RJ has been shown to contain a substantial number of extracellular vesicles (EVs) and in this study, we aimed to investigate the extent of involvement of RJEVs in wound healing associated effects. Molecular analysis of RJEVs verified the presence of important conserved exosomal markers such as CD63 and syntenin, as well as cargo molecules MRJP1, defensin-1 and jellein-3. RJEV internalization analysis demonstrated the involvement of membrane fusion as well as macropinocytosis or clathrin-dependent endocytosis into mammalian cells. Furthermore, RJEVs have demonstrated to modulate MSCs differentiation and secretome, as well as decrease LPS-induced inflammation in RAW 264.7 macrophages by blocking the MAPK pathway. In vivo studies confirmed anti-bacterial effects of RJEVs, and demonstrated an acceleration of wound healing in a splinted mouse model. Summarizing, this study suggests that RJEVs of potentially exosomal origin play a crucial role in the known effects of RJ by modulating the inflammatory phase and cellular response in wound healing.