Remodeling of energy-storing white fat into energy-consuming beige fat has led to a promising new approach to alleviate adiposity. Several studies have shown adipokines can induce white adipose tissues (WAT) beiging through autocrine or paracrine actions. Betatrophin, a novel adipokine, has been linked to energy expenditure and lipolysis but not clearly clarified. Here, we using high-fat diet induced obesity to determine how betatrophin modulate beiging and adiposity. We found that betatrophin-knockdown mice displayed less white fat mass and decreased plasma TG and NEFA levels. Consistently, inhibition of betatrophin leads to the phenotype change of adipocytes characterized by increased mitochondria contents, beige adipocytes and mitochondrial biogenesis-specific markers both in vivo and in vitro. Notably, blocking AMP-activated protein kinase (AMPK) signaling pathway is able to abolish enhanced beige-like characteristics in betatrophin-knockdown adipocytes. Collectively, down-regulation of betatrophin induces beiging in white adipocytes through activation of AMPK signaling pathway. These processes suggest betatrophin as a latent therapeutic target for obesity.