Extracellular vesicles (EVs) are known to impact microbial metabolic activities, but their specific role in Mn reduction remains arcane. We aim to uncover the underlying mechanisms by which EVs facilitate this process. EVs from Shewanella oneidensis MR-1, the model strain for extracellular electron transfer research, were extracted and 4D-FastDIA proteomics was utilized to identify EV-associated proteins and bacterial proteins.