Increased activity of intracellular glucocorticoid reactivating enzyme, 11-hydroxysteroid dehydrogenase type 1 (11-HSD1) in obese adipose tissue contributes to adipose dysfunction. As recent studies have highlighted a potential role of preadipocytes in adipose dysfunction, we tested the hypothesis that a variety of metabolic stress mediated by ceramide or AMP-activated protein kinase (AMPK) would regulate 11-HSD1 in preadipocytes. The present study is the first to show that 1) expression of 11-HSD1 in 3T3-L1 preadipocytes was robustly induced when cells were treated with cell-permeable ceramide analogue C₂ ceramide, bacterial sphingomyelinase and sphingosine 1-phosphate, 2) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-induced activation of AMPK augmented the expression and enzyme activity of 11-HSD1, and 3) these results were reproduced in human preadipocytes. We demonstrate for the first time that C₂ ceramide and AICAR markedly induced the expression of CCAAT/enhancer-binding protein (C/EBP) and its binding to 11-HSD1 promoter. Transient knockdown of C/EBP protein by small interfering RNA markedly attenuated the expression of 11-HSD1 induced by C₂ ceramide or AICAR. The present study provides novel evidence that ceramide- and AMPK-mediated signaling pathways augment the expression and activity of 11-HSD1 in preadipocytes by way of C/EBP, thereby highlighting a novel, metabolic stress-related regulation of 11-HSD1 in a cell-specific manner.