MicroRNAs are implicated as crucial mediators in metabolic diseases including obesity, diabetes, and non-alcoholic fatty liver diseases (NAFLD). Here, we show miR-27a attenuated hepatic de novo lipogenesis and alleviated obesity-initiated NAFLD through inhibiting Fasn and Scd1 in liver. Hepatic levels of miR-27a were significantly augmented in HFD-fed and ob/ob mice. Further studies demonstrated that miR-27a directly interacted with 3' untranslated region (3'-UTR) of hepatic Fasn and Scd1 mRNAs and reduced their expression levels in mice. Adenovirus-mediated overexpression of miR-27a robustly blocked sodium oleate-induced triglyceride (TG) accumulation in mouse primary hepatocytes and reduced liver TG contents in mice via repressing hepatic lipogenesis. Furthermore, ectopic expression of hepatic miR-27a impaired lipid contents of livers and attenuated NAFLD development through suppressing lipogenesis in HCD-fed and ob/ob mice. Together, our results reveal a critical role of miR-27a in lipid homeostasis of liver and pathogenesis of NAFLD.