Objectives The current study tested the hypothesis that glucose utilization differs between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), and investigated potential mechanisms for such a finding. Background VAT burden correlates better with cardiovascular risk than does SAT burden. Beyond volumetric measurement, glucose uptake in adipose tissue (AT) might reflect metabolic activity and provide pathophysiologic insight and aid risk stratification. Methods We retrospectively studied tissue-specific glucose uptake in vivo in clinically obtained whole-body fluorodeoxyglucose positron emission tomography (FDG-PET) scans in humans. We also assessed glucose uptake in vitro, using stromal vascular cells isolated from SAT and VAT of diet-induced obese C57BL/6 mice. Quantitative polymerase chain reaction (PCR) evaluated the expression of multiple genes involved in cellular glucose metabolism, including glucose transporters (GLUT-1, -3, and -4) and hexokinases (HK-1 and -2) in SAT and VAT of obese C57BL/6 mice. Results We analyzed whole-body FDG-PET scans from 31 obese and 26 lean patients. VAT exhibited higher FDG uptake compared with SAT (p < 0.0001) independent of age, sex, body mass index, comorbidities, and medications. To investigate mechanisms underlying this observation, we studied glucose uptake in the stromal vascular cell fraction of AT, which is rich in inflammatory cells. Stromal vascular cells from VAT of diet-induced obese C57BL/6 mice exhibited higher glucose uptake than those from SAT (p = 0.01). Evaluation of expression of glucose transporters (GLUT-1, -3, and -4) and hexokinases (HK-1 and -2), revealed increased expression of HK-1 in VAT-derived compared with SAT-derived stromal vascular cells, and also in visceral versus subcutaneous unfractionated AT. Conclusions In humans in vivo, VAT has increased glucose uptake compared with SAT, as determined noninvasively with FDG PET imaging. Differential stromal metabolic activity may be 1 mechanism underlying differences in metabolic activity of visceral and subcutaneous AT.