Glucocorticoid (GC) excess promotes visceral obesity and cardiovascular disease. Similar features are found in the highly prevalent metabolic syndrome in the absence of high levels of systemic cortisol. Whilst elevated activity of the glucocorticoid amplifying enzyme 11 Hydroxysteroid Dehydrogenase type 1 (11-HSD1) within adipocytes might explain this paradox, the potential role of 11-HSD1 in preadipocytes is less clear; human omental adipose stromal vascular (ASV) cells exhibit 11-dehydrogenase activity (inactivation of GCs) probably due to the absence of co-factor provision by hexose-6-phosphate dehydrogenase (H6PDH). To clarify the depot-specific impact of 11-HSD1, we assessed whether preadipocytes in ASV from mesenteric (as a representative of visceral adipose tissue) and subcutaneous displayed 11-HSD1 activity in mice. 11-HSD1 was highly expressed in freshly isolated ASV cells, predominantly in preadipocytes. 11-HSD1 mRNA and protein levels were comparable between ASV and adipocyte fractions in both depots. 11-HSD1 was an 11-reductase, thus reactivating glucocorticoids in ASV cells, consistent with H6PDH mRNA expression. Unexpectedly, glucocorticoid reactivation was higher in intact mesenteric ASV despite a lower expression of 11-HSD1 mRNA and protein (homogenate activity) levels than subcutaneous ASV. This suggests a novel depot-specific control over 11-HSD1 enzyme activity. In vivo, high fat (HF) diet-induced obesity was accompanied by increased visceral fat preadipocyte differentiation in wild-type but not 11-HSD1^-/-mice. The results suggest that 11-HSD1 reductase activity is augmented in mouse mesenteric preadipocytes where it promotes preadipocyte differentiation and contributes to visceral fat accumulation in obesity.