The CCAAT/enhancer-binding protein beta (C/EBPbeta) is required for adipocyte differentiation and maturation. We have studied the role of the transcription factor, C/EBPbeta, in the development of diet-induced obesity. Mice with a deletion in the gene for C/EBPbeta (C/EBPbeta(-/-)) and wild-type mice were fed a high-fat diet (60% fat) for 12 weeks. The C/EBPbeta(-/-) mice lost body fat, whereas the wild-type mice increased their total body fat on a high-fat diet. The C/EBPbeta(-/-) mice had lower levels of blood triglycerides, free fatty acids, cholesterol, and hepatic triglyceride accumulation compared with the wild-type mice, thus protecting them from diet-induced obesity and fatty liver on a high-fat diet. Deletion of C/EBPbeta gene resulted in greatly reducing hepatic lipogenic genes, acetyl CoA carboxylase, and fatty acid synthase and increasing the expression of beta-oxidation genes in the brown adipose tissue. CO(2) production was significantly higher in the C/EBPbeta(-/-) mice as was the level of uncoupling protein (UCP)-1 and UCP-3 in the muscle. In conclusion, the transcription factor C/EBPbeta is an important regulator in controlling lipid metabolism and in the development of diet-induced obesity.