We investigated the effect of myostatin on the differentiation of bovine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from perirenal adipose tissue of approximately 30-month-old Japanese Black steers. After confluence, the differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin, and troglitasone for 2 days, and then subsequently cultured for 6 days. The cells were treated with myostatin during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. We measured the terminal differentiation markers such as glycerol-3-phosphate dehydrogenase activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein mRNA at the end of cultures. The treatment with myostatin throughout the differentiation period severely suppressed the induction of all differentiation markers. The treatment with myostatin in the early phase of differentiation also suppressed the induction of terminal differentiation markers but three-fold higher dose of myostatin was required for the suppression compared with its treatment throughout the differentiation period. Myostatin treatment reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma mRNA and interfered with the induction of CCAAT/enhancer binding protein (C/EBP) alpha mRNA. We also observed that follistatin stimulates preadipocyte differentiation in the presence of myostatin. These results suggest that myostatin inhibits bovine preadiopocyte differentiation through suppressing PPARgamma and C/EBPalpha mRNA expressions and that follistatin counteracts the suppressive effect of myostatin.