Human adipose tissue (AT) is well recognized as a key player in energy homeostasis due to its storage function of circulating non-esterified free fatty acids (NEFA) as triglycerides in adipocytes. A defective extraction of plasma NEFA by adipose tissue might lead to post-prandial hyperlipidemia and thus increase the risk to develop insulin resistance. Human AT-endothelial cells (ECs) are the first cell type that modulates lipid passage from the blood compartment to the adipose tissue. However, mechanisms underlying lipid handling in human adipose tissue remain to be fully characterized in AT-ECs. Using a fluorescent-activated cell sorter (FACS) approach, uptake of fluorescent labelled fatty acid by stroma-vascular cells from human subcutaneous AT was studied. AT-ECs (CD34+/CD31+ cells) are able to incorporate several saturated FA such as palmitic (C16:0) and lauric (C12:0) acids. FA-uptake kinetics of AT-ECs depend on the length of fatty acid chain and differ from that of progenitors cells (CD34+/CD31- cells). Trans-endothelial transport of NEFA in human adipose tissue may occur by diffusion or may be mediated by plasma-membrane and cytoplasm proteins. Real-time PCR and FACS analyses performed on AT-ECs isolated from freshly harvested stroma-vascular fraction revealed that these cells express several fatty acid transport proteins (FATPs) and fatty acid binding proteins (FABPs) and the fatty acid translocase FAT/CD36. AT-ECs also express the transcription factor PPARγ known as the major regulator of adipocyte lipid storage and we observed that maintaining AT-ECs in culture with an adipogenic media supplemented by PPARγ agonist leads to lipid droplet formation. This effect of PPARγ agonists was only observed on endothelial cells isolated from the adipose tissue. In agreement with lipid droplet formation, immunohistochemical approaches revealed a marked accumulation of the lipid droplet-associated protein ADRP/Adipophilin in endothelial cells of subcutaneous AT from obese patients (BMI>30) (n=8) compared to lean patient (BMI<25) (n=7). In addition, compared to AT-ECs of non obese patients (n=4-6), AT-EC of obese patients (n=4-9) exhibited a lower expression of proteins involved in the endothelial barrier function including VE-cadherin (adherens junction), JAM2 and Claudin 5 (thight junction) and CD31. Our results showed that endothelial cells of human adipose tissue exhibit specific properties regarding lipid handling and suggest that, during obesity, a local dysfunction of adipose tissue endothelial cells occurs. This dysfunction might be a primary event involved in the genesis of obesity-associated pathologies.