Adiponectin, a hormone secreted from adipocytes and released at a high rate into the circulation, plays a pivotal role in maintaining insulin sensitivity at the whole-body level. Despite the importance of this adipokine in metabolic homoeostasis, the mechanism of its secretion from adipocytes remains largely unclear. In the present study, we investigated the subcellular localization of adiponectin, and its secretion regulation in 3T3-L1-differentiated adipocytes, using biochemical methods and fluorescence microscopic imaging. We show that adiponectin is localized in vesicular compartments with no apparent overlap with the Golgi apparatus or endosomes. Moreover, adiponectin-containing vesicles are enriched in two distinct pools: one at the plasma membrane (PM) and the other co-fractionating with endoplasmic reticulum membranes. When viewed under a total internal refection fluorescence microscope, a subset of adiponectin-Venus vesicles is readily observed in proximity to PMs, and could be released in response to insulin. Insulin-stimulated adiponectin release appears to be from a pre-existing pool of vesicles, and is not dependent on new protein synthesis, because adiponectin mRNA levels remain unchanged over a 6-h period of insulin treatment, and inhibition of protein synthesis has no effect on adiponectin release. Disruption of insulin signalling by inhibitors of phosphoinositide 3-kinase and protein kinase B (Akt)-1/2 abrogates the stimulated release of adiponectin. Taken together, our results show that adiponectin is stored in a unique vesicular compartment, and released through a regulated exocytosis pathway that is dependent on insulin signalling.