Connective tissue growth factor (CTGF), also known as CCN-2, is a cysteine-rich secreted protein that is involved in a range of biological processes including regulation of cell growth and differentiation. Our previous in vitro studies have shown that CCN-2 inhibits adipocyte differentiation, although whether CCN-2 is regulated in vivo in adipogenesis is undetermined and was investigated in this study. C57BL/6 male mice were fed either standard laboratory chow (ND) or diet high in fat (HFD, 45% fat) for 15 or 24 weeks. HFD animals which gained >5 g in weight (termed HFD-Fat) were insulin resistant and were compared to HFD fed animals which failed to gain weight (termed HFD-Lean). HFD-Fat mice had significantly increased CCN-2 mRNA levels in both the subcutaneous and epididymal fat pads, whereas CCN-2 mRNA was not induced in the epididymal site in HFD-Lean mice. Also in HFD-fed animals, epididymal CCN-2 mRNA correlated positively with key genes involved in adipocyte differentiation, adiponectin and PPAR (p<0.001 and p<0.002, respectively). Additionally, epididymal CCN-2 mRNA correlated positively with two markers of tissue turnover, PAI-1 in HFD-Fat mice only, and TIMP-1 but only in the HFD-Lean mice. Collectively, these findings suggest that CCN-2 plays a role in adipocyte differentiation in vivo and thus in the pathogenesis of obesity linked with insulin resistance.