Adipogenesis is regulated by a complicated network of transcription factors among which PPAR gamma and C/EBP family members are the major regulators. During adipogenesis, C/EBP beta is induced early and then transactivates PPAR gamma and C/EBP alpha, which cooperatively induce genes whose expressions give rise to the mature adipocyte phenotype. Identifying the factors that influence the expression and activity of C/EBP beta should provide additional insight into the mechanisms regulating adipogenesis. Here, we demonstrate that depletion of Ajuba in 3T3-L1 cells significantly decreases mRNA and protein levels of PPAR gamma and C/EBP alpha and impairs adipocyte differentiation, while overexpression increases expression of these genes and promotes adipocyte differentiation. Moreover, restoration of C/EBP alpha or PPAR gamma expression in Ajuba-deficient 3T3-L1 cells improves the impaired lipid accumulation. Mechanistically, Ajuba interacts with C/EBP beta and recruits CBP to facilitate the binding of C/EBP beta to the promoter of PPAR gamma and C/EBP alpha, resulting in increased H3 histone acetylation and target gene expression. Collectively, these data indicate that Ajuba functions as a co-activator of C/EBP beta, and may be an important therapeutic target for combating obesity-related diseases.