Bacterial extracellular vesicles (EVs) are vesicles secreted by bacteria into the extracellular environment. Containing DNA, RNA and proteins, EVs are implicated to mediate intercellular communications. The marine cyanobacterium , the most abundant photosynthetic organism in marine ecosystems, has been shown to generate EVs continuously during cell growth. However, biogenesis and functions of EVs released by remain largely unclear. Here, we isolated and characterized EVs released by MED4 culture. We found that the majority of MED4 EVs are elliptical and enriched with specific proteins performing particular cellular functions. The light-dark cycle has been demonstrated to affect the cell cycle of , with cell division occurring at night time. Interestingly, we found that the net production of MED4 EVs was faster during the night time. Moreover, we revealed that MED4 EVs that are released or absorbed in the night time are enriched with distinct proteins, suggesting the release and absorbance of EVs are influenced by the diel cycle. We found that inhibiting cell division decreased the net production of MED4 EVs during the night time, suggesting that cell division is important for the biogenesis of MED4 EVs. These analyses provide novel insights into biogenesis and functions of EVs released from bacteria.This article is part of the Theo Murphy meeting issue 'Circadian rhythms in infection and immunity'.