Selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) have considerable potential as a treatment for metabolic syndrome including type 2 diabetes mellitus and obesity. To identify 11β-HSD1 inhibitors, we conducted high-throughput screening (HTS) of active natural product extracts from the Korea Chemical Bank, including Tanshinone I, Tanshinone IIA, and flavanone derivatives, and 2- and 3-phenyl-4H-chromen-4-one. Then Tanshinone IIA and its derivatives were targeted for the development of a lead compound according to the HTS results. However, the mechanism for anti-adipogenic effect through 11β-HSD1 enzyme inhibition by Tanshinone IIA is not clear. Tanshinone IIA (2a) concentration-dependently inhibited 11β-HSD1 activity in human and mouse 11β-HSD1 overexpressed cells and 3T3-L1 adipocytes. Tanshinone IIA (2a) also inhibited 11β-HSD1 enzyme activities in murine liver and fats. Furthermore, Tanshinone IIA (2a)-suppressed adipocyte differentiation of cortisone-induced adipogenesis in 3T3-L1 cells was associated with the suppression of the cortisone-induced adipogenesis-specific markers mRNA and protein expression. In 3T3-L1 preadipocytes, Tanshinone IIA (2a)-inhibited cortisone induced reactive oxygen species formation in a concentration-dependent manner. Thus, these results support the therapeutic potential of Tanshinone IIA (2a) as a 11β-HSD1 inhibitor in metabolic syndrome patients.