Myostatin (MSTN) is a secreted growth inhibitor expressed in muscle and adipose. We sought to determine whether expression of MSTN, its receptor activin RIIb (ActRIIb), or its binding protein follisatin-like-3 (FSTL3) is altered in subcutaneous (SQF) or visceral (VF) adipose or in skeletal muscle in response to obesity. MSTN and ActRIIb mRNA levels were low in SQF and VF from wild type mice but were 50-100-fold higher in both SQF and VF from ob/ob compared to wild type mice. FSTL3 mRNA levels were increased in SQF but decreased in VF in ob/ob compared to wild type mice. Moreover, MSTN mRNA levels were 2-fold greater in tibialis anterior (TA) from ob/ob mice, while ActRIIb and FSTL3 mRNA levels were unchanged. MSTN mRNA levels were also increased in TA and SQF from mice on a high fat diet. Injection of ob/ob mice with recombinant leptin caused FSTL3 mRNA levels to decrease in both VF and SQF in ob/ob mice; MSTN and ActRIIb mRNA levels tended to decrease only in VF. Finally, MSTN mRNA levels and promoter activity were low in adipogenic 3T3-L1 cells, but a MSTN promoter-reporter construct was activated in 3T3-L1 cells by co-transfection with the adipogenic transcription factors SREBP1c, C/EBP-, and PPAR-. These results demonstrate that expression of MSTN and its associated binding proteins can be modulated in adipose tissue and skeletal muscle by chronic obesity, and suggest that alterations in their expression may contribute to the changes in growth and metabolism of lean and fat tissues occurring during obesity.