Objective: Brown adipocytes play important roles in the regulation of energy homeostasis by uncoupling protein 1-mediated non-shivering thermogenesis. Recent studies suggest that brown adipocytes as novel therapeutic targets for combating obesity and associated diseases, such as type II diabetes. However, the molecular mechanisms underlying brown adipocyte differentiation and function are not fully understood. Methods: We employed previous findings obtained through proteomic studies performed to assess proteins displaying altered levels during brown adipocyte differentiation. Here, we performed assays to determine the functional significance of their altered levels during brown adipogenesis and development. Results: We identified isocitrate dehydrogenase 1 (IDH1) as upregulated during brown adipocyte differentiation, with subsequent investigations revealing that ectopic expression of IDH1 inhibited brown adipogenesis, whereas suppression of IDH1 levels promoted differentiation of brown adipocytes. Additionally, Idh1 overexpression resulted in increased levels of intracellular alpha-ketoglutarate (alpha-KG) and inhibited the expression of genes involved in brown adipogenesis. Exogenous treatment with alpha-KG reduced brown adipogenesis during the early phase of differentiation, and ChIP analysis revealed that IDH1-mediated alpha-KG reduced trimethylation of histone H3 lysine 4 in the promoters of genes associated with brown adipogenesis. Furthermore, administration of alpha-KG decreased adipogenic gene expression by modulating histone methylation in brown adipose tissues of mice. Conclusion: These results suggested that the IDH1-alpha-KG axis plays an important role in regulating brown adipocyte differentiation and might represent a therapeutic target for treating metabolic diseases.