Rhein (RH), a compound purified from Radix et Rhizoma Rhei, has been used to alleviate liver and kidney damage. It is found that RH inhibited the differentiation of 3T3-L1 preadipocytes induced by differentiation medium in a time- and dose-dependent manner. It was revealed that RH downregulated the expression of adipogenesis-specific transcription factors PPARγ and C/EBPα, as well as their upstream regulator, C/EBPβ. Furthermore, the PPARγ target genes that are involved in adipocyte differentiation, such as CD36, aP2, acyl CoA oxidase, uncoupled protein 2, acetyl-CoA carboxylase, and fatty acid synthase, were reduced after to RH. In addition, high-fat diet-induced weight gain and adiposity were reversed by RH in C57BL/6 mice. Consistent with the cells' results, RH downregulated the mRNA levels of PPARγ and C/EBPα, and their downstream target genes in C57BL/6 mice. Taken together, adipocyte differentiation and adipogenesis were inhibited by RH in cultured cells and in rodent models of obesity. The evidence implied that RH was a potential candidate for preventing metabolic disorders.