CCAAT/enhancer binding protein α (C/EBPα) is a critical factor in glucose metabolism in the neonate as revealed by conventional C/EBPα-null mice that do not survive beyond the first day after birth because of severe hypoglycemia and a deficiency in hepatic glycogen accumulation. To elucidate the function of C/EBPα in leptin-deficient mouse (ob/ob) liver, a C/EBPα-liver null mouse on an ob/ob background (ob/ob-C/EBPα/Cre+) was produced using a floxed C/EBPα allele and Cre recombinase under control of the albumin promoter (AlbCre). The C/EBPα-deficient liver in ob/ob mice had significantly decreased triglyceride content compared with equivalent mice lacking the AlbCre transgene (ob/ob-C/EBPα/Cre-). Expression of genes involved in lipogenesis including fatty acid synthase, acetyl-coenzyme A carboxylase, stearoylcoenzyme A desaturase 1 and ATP-citrate lyase dramatically decreased in ob/ob-C/EBPα/Cre+ mouse liver. Induction of these lipogenic genes by a high-carbohydrate diet caused an exacerbation in the development of fatty liver and an increase in liver size, hepatic triglyceride, and cholesterol contents in ob/ob-C/EBPα/Cre- mice but not in ob/ob-C/EBPα/Cre+ mice. Deficiency in hepatic C/EBPα expression caused an exacerbation of hyperglycemia because of decreased insulin secretion. Taken together, these results indicate that hepatic C/EBPα plays a critical role in the acceleration of lipogenesis in ob/ob mice and in glucose homeostasis by the indirect regulation of insulin secretion.