Extracellular vesicles (EVs) are vesicles released by cells that carry proteins, nucleic acids and lipids. They have been widely described as important for cell-to-cell communication, causing physiological changes in recipient cells. Despite the increase of their study, molecular mechanisms responsible of their biogenesis and release are still largely unknown. We previously reported that hypoxia increases the release of EVs in adipose cells. On the other hand, autophagy is a cellular homeostasis maintenance response also activated upon low oxygen conditions. Aim: To determine whether hypoxia and autophagy increase EVs release. Methods: Several cell lines (HEK293, 3T3 fibroblasts and SK1-N neuroblastoma cells) were exposed to normoxic or hypoxic conditions (1% O2) for different periods of time. Autophagy was induced by nutrient starvation with Hank’s Balanced Salt Solution (HBSS). EVs release profile and autophagy activation were determined in control normoxic, hypoxia exposed or nutrient starved cells. Autophagy progression was independently verified by over expression of an autophagy-related protein: TP53INP2. EVs were isolated from culture media supernatants by differential ultracentrifugation and quantified by Nanoparticle Tracking analysis. Induction of autophagy was confirmed by western blot of total cellular lysates. Data was statistically analysed running One-way ANOVA tests using GraphPad Prism 6. Results: Hypoxia induced EVs release upon prolonged hypoxic stimulus and peaked at a time coincident with activation of autophagy response. Starvation-induced autophagy showed an increase on EVs release parallel to autophagy progression. Enhanced EVs release was also confirmed following the overexpression of TP53INP2. Conclusion: Our results suggest that autophagy increases EVs release and that this may be one of the molecular mechanisms involved in EVs release under low oxygen conditions.