Background and aims: Exosomes and microvesicles are extracellular vesicles (EVs) shed by many cell types that mediate cell to cell communication. EVs contain proteins and RNA species that modulate biochemical responses in target cells. Adipocytes have been shown to produce extracellular vesicles. During obesity adipocytes are subject to hypoxia. The aim of our study was to investigate whether EVs released by hypoxic adipocytes have autocrine effects in regulating insulin action and glucose uptake. Methods: We isolated EVs from the conditioned media of differentiated 3T3L1 adipocytes cultured under normoxia (control cells) or from cells exposed to hypoxia (1% O2) for 24hrs. EVs were purified from the culturing media using centrifugation techniques and characterized using biophysical and biochemical methods, including nanoparticle tracking analysis, cryoelectron microscopy, and immunoblotting techniques. EVs were then tested for their ability to modulate insulin action and insulin-stimulated glucose uptake in 3T3L1 adipocytes. Independent sets of differentiated 3T3L1 adipocytes were either left untreated or treated with EVs purified from control or hypoxic adipocytes for 24hr and subsequently their response to insulin stimulation was evaluated by western blotting and glucose uptake assays. Results: We found that 3T3L1 adipocytes release EVs of heterogeneous sizes as determined by cryoelectron microscopy and nanoparticle tracking analysis. Hypoxia increased the release of EVs by adipocytes. Immunoblotting analysis showed that EV preparations were enriched in exosomal markers and markedly devoid of other organelle markers. Adipocytes treated with EVs obtained from hypoxic cells displayed a reduced insulin-mediated activation of glucose uptake compared to those left untreated or treated with EVs derived from control adipocytes. No differences in the expression levels of the glucose transporter Glut4 were observed in cells treated with EVs from hypoxic adipocytes compared to those left untreated or treated with EVs from control cells. However, cells treated with EVs released from hypoxic cells exhibited reduced insulin-mediated activation of phosphatidylinositol 3-kinase as seen by a reduced phosphorylation of the downstream kinase AKT, without alterations in the total AKT levels. Conclusions: Our data suggest that EVs released by hypoxic adipocytes contribute to insulin resistance, as they impair insulin signalling and glucose uptake in adipose cells.