Background: Adaptation of fat depots to change in fuel availability is critical for metabolic flexibility and cardiometabolic health. The mechanisms responsible for fat depot-specific lipid sensing and shuttling remain elusive. Adipose tissue microvascular endothelial cells (AT-EC) regulates bidirectional fatty acid fluxes depending on fed or fasted state. How AT-EC sense and adapt to metabolic changes according to AT location remains to be established. Methods: We combined transcriptional analysis of native human AT-EC together with in vitro approaches in primary human AT-EC and in vivo and ex vivo studies of mice under fed and fasted conditions. Results: Transcriptional large-scale analysis of human AT-EC isolated from gluteofemoral and abdominal subcutaneous AT revealed that the endothelium exhibits a fat depot–specific signature associated with lipid handling and Notch signaling enrichment. We uncovered a functional link between metabolic status and endothelial DLL4 (delta-like canonical notch ligand 4), which decreases with fasting. DLL4 regulates fatty acid uptake through nontranscriptional modulation of macropinocytosis-dependent long chain fatty acid uptake. Importantly, the changes in DLL4 expression, in response to energy transition state, is impaired under obesogenic conditions, an early alteration coinciding with a defect in systemic fatty acid fluxes adaptation and a resistance to weight loss. Conclusions: DLL4 is a major actor in the adaptive mechanisms of AT-EC to regulate lipid fluxes. It likely contributes to fat depot-dependent metabolism in response to energy transition states. AT-EC alteration with obesity may favor metabolic inflexibility and the development of cardiometabolic disorders.Read less <