The adipocyte enzyme 11-hydroxysteroid dehydrogenase type 1 (11-HSD1) amplifies local glucocorticoid action by generating active glucocorticoids from inactive metabolites, and has emerged as a key player in the pathogenesis of central obesity and metabolic syndrome. However, the regulation of adipocyte 11-HSD1 is incompletely understood. Therefore, the present study was designed to investigate the effects of insulin and glucocorticoid as well as their underlying molecular mechanisms on 11-HSD1 activity and expression in 3T3-L1 adipocytes, and determine if the in vitro findings could be confirmed in vivo. Our main in vitro findings are (a) insulin stimulated while dexamethasone inhibited 11-HSD1 activity and expression in a time- and concentration-dependent manner; (b) the effect of dexamethasone was mimicked by both cortisol and corticosterone, but blocked by the glucocorticoid receptor (GR) antagonist RU486; (c) the p38 MAPK inhibitor SB220025, but not the ERK inhibitor U0126 or the PI3 kinase inhibitor LY294002, prevented insulin-stimulation of 11-HSD1 activity; and (d) while dexamethasone did not alter the half-life of 11-HSD1 mRNA, insulin doubled it. Taken together, these in vitro results demonstrate that insulin stimulates adipocyte 11-HSD1 through a post-transcriptional mechanism that involves activation of the p38 MAPK signaling pathway, while dexamethasone exerts an opposite effect by a GR-mediated transcriptional mechanism. In contrast, both insulin and dexamethasone augmented 11-HSD1 activity and expression in rat white adipose tissue in vivo, thus confirming the role of insulin but revealing a fundamental difference regarding the role of dexamethasone in regulating adipocyte 11-HSD1 between the two model systems.