Adiponectin is a fat cell-derived hormone with insulin-sensitizing properties. Low plasma adiponectin levels are associated with insulin resistance as found in obesity. One of the mechanisms for this finding is hampered insulin signaling via phosphatidylinositol 3-kinase (PI3K) with concomitant decreased adiponectin secretion. Because insulin can also stimulate signaling at the level of mammalian target of rapamycin (mTOR) by a mechanism that is dependent on the presence of amino acids, the role of mTOR signaling in adiponectin secretion was studied. In view of the vesicular nature of adiponectin secretion, the role of lysosomes was explored as well. In 3T3-L1 adipocytes, both insulin and amino acids stimulated adiponectin secretion. The stimulation by insulin was PI3K dependent but mTOR independent. The stimulation by amino acids was independent of both PI3K and mTOR. Whereas the effect of insulin via PI3K was mainly on adiponectin secretion from adipocytes, the effect of amino acids was predominantly due to their role as substrates for adiponectin synthesis. The acidotropic agents ammonia and methylamine, but not the lysosomal protease inhibitor leupeptin and the autophagy inhibitor 3-methyladenine, strongly inhibited adiponectin secretion and increased the intracellular adiponectin pool. In conclusion, adiponectin production is substrate driven. Phosphatidylinositol 3-kinase and an acidic lysosomal pH, but not amino acid-mediated mTOR signaling or lysosomal breakdown, are involved in adiponectin secretion.