ABSTRACTBacterial cells form and release membrane vesicles (MVs) originating from cellular membranes. In recent years, many biological functions of bacterial MVs have been identified. Here, we show that MVs derived fromCorynebacterium glutamicum, a model organism for mycolic acid-containing bacteria, can mediate iron acquisition and other phylogenetically related bacteria. Lipid/protein analysis and iron quantification assay indicate thatC. glutamicumMVs formed by outer mycomembrane blebbing can load ferric iron (Fe3+) as its cargo. Iron-loadedC. glutamicumMVs promoted the growth of producer bacteria in iron-limited liquid media. MVs were received byC. glutamicumcells, suggesting a direct transfer of iron to the recipient cells. Cross-feeding ofC. glutamicumMVs with phylogenetically close (Mycobacterium smegmatisandRhodococcus erythropolis) or distant (Bacillus subtilis) bacteria indicated thatC. glutamicumMVs could be received by the different species tested, while iron uptake is limited toM. smegmatisandR. erythropolis. In addition, our results indicate that iron loading on MVs inC. glutamicumdoes not depend on membrane-associated proteins or siderophores, which is different from what has been shown in other mycobacterial species. Our findings illustrate the biological importance of MV-associated extracellular iron forC. glutamicumgrowth and suggest its ecological impact on selected members of microbial communities.IMPORTANCEIron is an essential element of life. Many bacteria have developed iron acquisition systems, such as siderophores, for external iron uptake.Corynebacterium glutamicum, a soil bacterium known for its potential for industrial applications, was shown to lack the ability to produce extracellular, low-molecular-weight iron carriers, and it remains elusive how this bacterium acquires iron. Here, we demonstrated that MVs released fromC. glutamicumcells could act as extracellular iron carriers that mediate iron uptake. Although MV-associated proteins or siderophores have been shown to play critical roles in MV-mediated iron uptake by other mycobacterial species, the iron delivery throughC. glutamicumMVs is not dependent on these factors. Moreover, our results suggest that there is an unidentified mechanism that determines the species specificity of MV-mediated iron acquisition. Our results further demonstrated the important role of MV-associated iron.