TRB3 has been implicated in the regulation of several biological processes in mammalian cells through its ability to influence Akt and other signaling pathways. In this study, we investigated the role of TRB3 in regulating adipogenesis and the activity of adipogenic transcription factors. We find that TRB3 is expressed in 3T3-L1 preadipocytes, and this expression is transiently suppressed during the initial days of differentiation concomitant with induction of C/EBPbeta. This event appears to be a prerequisite for adipogenesis. Overexpression of TRB3 blocks differentiation of 3T3-L1 cells at a step downstream of C/EBPbeta. Ectopic expression of TRB3 in mouse fibroblasts also inhibits the C/EBPbeta-dependent induction of PPARgamma2 and blocks their differentiation into adipocytes. This inhibition of preadipocyte differentiation by TRB3 appears to be the result of two complementary effects. First, TRB3 inhibits extracellular signal-regulated kinase activity, which prevents the phosphorylation of regulatory sites on C/EBPbeta. Second, TRB3 directly interacts with the DR1 domain of C/EBPbeta in the nucleus, further inhibiting both its ability to bind its response element and its ability to transactivate the C/EBPalpha and a-FABP promoters. Thus, TRB3 is an important negative regulator of adipogenesis that acts at an early step in the differentiation cascade to block the C/EBPbeta proadipogenic function.