Inside-out: unpredicted Annexin A2 localisation on the surface of extracellular vesicles Extracellular vesicles (EVs) contain many proteins, both cytosolic and surface bound. The current model for EV biogenesis dictates that cytosolic proteins remain in the lumen and cell surface proteins reside on the outside of vesicles. This is consistent with the traditional protein trafficking pathway, where proteins destined for the plasma membrane contain a signal sequence targeting them to the secretory pathway. According to this ‘classical’ pathway for membrane and secretory protein trafficking, proteins lacking a signal sequence should not reside at the cell surface. It has been shown that transmembrane proteins are retained in the membrane of EVs and RNAs reside in the lumen of EVs. However, there is little known about the packaging and location of other proteins enriched in EVs. Annexin A2 is a cytosolic protein abundant in EVs. We show for the first time that Annexin A2 is expressed not only in the lumen of EVs as predicted but also on the surface of EVs. This raises fundamental questions regarding Annexin A2 transport to the outer leaflet of the EV membrane as it lacks a signal peptide for secretion.