MicroRNAs (miRNAs) are involved in a plethora of important biological processes, from embryonic development to homeostasis in adult tissues. Recently, miRNAs have emerged as a class of epigenetic regulators of metabolism and energy homeostasis. We have investigated the role of miRNAs in the regulation of adipogenic differentiation. In this article, we demonstrate that the miR-27 gene family is downregulated during adipogenic differentiation. Overexpression of miR-27 specifically inhibited adipocyte formation, without affecting myogenic differentiation. We also found that expression of miR-27 resulted in blockade of expression of PPARγ and C/EBPα, the two master regulators of adipogenesis. Importantly, expression of miR-27 was increased in fat tissue of obese mice and was regulated by hypoxia, an important extracellular stress associated with obesity. Our data strongly suggest that miR-27 represents a new class of adipogenic inhibitors and may play a role in the pathological development of obesity.