Non-alcoholic fatty liver disease (NAFLD) is a common liver disease, characterized by the excess accumulation of lipids in the liver. It has been demonstrated that the dysregulation of lipid droplet (LD)-associated proteins may be involved in the development of NAFLD. Adipose differentiation-related protein (ADRP), as one of the major LD-associated proteins, is expressed in normal and steatotic livers; however, the exact role of ADRP in the liver remains unknown. Previous studies have indicated that metformin, as an antidiabetic drug, effectively ameliorates NAFLD. However, its cellular and molecular mechanisms of action remain to be elucidated. Therefore, the aim of this study was to determine the role of ADRP in the metformin-mediated regulation of hepatic steatosis. We examined the effects of meformin in vivo and in vitro using ob/ob mice and primary hepatocytes, respectively. Lipid accumulation in the hepatocytes was induced by treatment with oleate. Our results revealed that metformin prevented hepatic steatosis in ob/ob mice and inhibited oleate-induced lipid accumulation in primary hepatocytes. Furthermore, using real-time PCR and western blot analysis, we examined the mRNA and protein expression of ADRP, respectively. We found that metformin significantly decreased the expression levels of ADRP. In addition, to further clarify the role of ADRP in lipid accumulation, we generated recombinant adenoviruses to induce the overexpression of ADRP and to knockdown ADRP. In the hepatocytes in which ADRP was overexpressed, the reducing effects of metformin on lipid accumulation were diminished. However, the knockdown of ADRP using siRNA targeting ADRP reduced the accumulation of triglycerides. Taken together, our data demonstrate that metformin prevents hepatic steatosis by regulating the expression of ADRP, which may be a key target in the treatment of NAFLD.