Abstract Control of fatty acid storage and release in adipose tissue is fundamental in energy homeostasis and the development of obesity and type 2 diabetes. We here take the whole signalling network into account to identify how insulin and β-adrenergic stimulation in concert controls lipolysis in mature subcutaneous adipocytes obtained from non-diabetic and, in parallel, type 2 diabetic women. We report that, and show how, the anti-lipolytic effect of insulin can be fully explained by protein kinase B (PKB/Akt)-dependent activation of the phosphodiesterase PDE3B. Through the same PKB-dependent pathway β-adrenergic receptor signalling, via cAMP and PI3Kα, is anti-lipolytic and inhibits its own stimulation of lipolysis by 50%. Through this pathway both insulin and β-adrenergic signalling control phosphorylation of FOXO1. The dose–response of lipolysis is bell-shaped, such that insulin is anti-lipolytic at low concentrations, but at higher concentrations of insulin lipolysis was increasingly restored due to inhibition of PDE3B. The control of lipolysis was not altered in adipocytes from diabetic individuals. However, the release of fatty acids was increased by 50% in diabetes due to reduced reesterification of lipolytically liberated fatty acids. In conclusion, our results reveal mechanisms of control by insulin and β-adrenergic stimulation — in human adipocytes — that define a network of checks and balances ensuring robust control to secure uninterrupted supply of fatty acids without reaching concentrations that put cellular integrity at risk. Moreover, our results define how selective insulin resistance leave lipolytic control by insulin unaltered in diabetes, while the fatty acid release is substantially increased.