OBJECTIVE: Brown adipose tissue (BAT) produces heat using chemical energy of lipids and glucose, a function induced by cold exposure or diet. The brown adipogenesis is tightly controlled in a coordinated interplay between several transcriptional factors. It is not known what enables and coordinates this robust program of concerted cooperation between the transcriptional factors and co-regulators necessary for the brown adipogenesis. MATERIALS/METHODS: A. In vivo studies--we investigated the expression levels of miR-27a and b in mice after cold exposure. B. Using gene expression and functional studies together with high throughput imaging in primary preadipocytes, and cell culture models, we investigated the role of miR-27 in beige and brown adipogenesis. C. Using gene silencing and rescue experiments we dissected the molecular mechanisms of the miR-27 action. RESULTS: After cold exposure, miR-27 is downregulated in BAT and subcutaneous white adipose tissue (SAT). MiR-27 is also downregulated during brown adipogenesis of primary preadipocytes in vitro, and it directly targets and negatively regulates the essential components of the brown transcriptional network: Prdm16, Pparα, Creb, and in part Pgc1β. Together with its direct effect on Pparγ, and indirect on Pgc1α, mir-27 decreases brown differentiation of cultured cells and of primary SAT preadipocytes. CONCLUSIONS: Our results point to miR-27 as a central upstream regulator of the transcriptional network involved in beige and brown adipogenesis after cold exposure, and suggest miR-27 inhibition as a novel therapeutic approach for metabolic diseases aiming at increasing the beige/brown fat mass.