Adipocyte differentiation is a key process implicated in the pathogenesis of obesity and insulin resistance. Its regulation is triggered by a cascade of transcription factors, including the CCAAT/enhancer binding proteins (C/EBPs) and PPAR. Growth factors such as transforming growth factor-1 (TGF-1) are known to inhibit adipocyte differentiation in vitro via the C/EBP pathway, and in vivo, but whether a downstream mediator of TGF-1, connective tissue growth factor (CTGF) also known as CCN2, has a similar role, is unknown. Mouse 3T3-L1 cells were differentiated into adipocytes using standard methods and effects and regulation of CTGF were studied. Intervention with rhCTGF during differing stages of differentiation caused an inhibition in the development of the adipocyte phenotype, according to the gene expression of the differentiation markers adiponectin and PPAR, as well as suppression of lipid accumulation and expression of the lipogenic enzyme, glycerol-3-phophate dehydrogenase. While CTGF gene expression promptly fell by 90% as 3T3-L1 preadipocytes differentiated into mature adipocytes, CTGF mRNA expression was induced by added TGF-1. CTGF applied to cells early in the course of differentiation inhibited total cell protein levels and nuclear localization of the isoform of C/EBP (C/EBP-) and subsequently, total cell C/EBP- levels. CTGF also inhibited the adipocyte differentiation program in primary cultures of mouse predipocytes. Expression of CTGF mRNA was two-fold higher in the central fat depots of mice compared with subcutaneous fat, suggesting a potential role for CTGF in vivo. In summary, these data show that CTGF inhibits the adipocyte differentiation program.