Follistatin is secreted from the liver and is involved in the regulation of muscle mass and insulin sensitivity via inhibition of activin A in humans. The secretion of follistatin seems to be stimulated by glucagon and inhibited by insulin, but only limited knowledge on the postprandial regulation of follistatin exists. Moreover, results on postoperative changes after Roux-en-Y gastric bypass (RYGB) are conflicting with reports of increased, unaltered and lowered fasting concentrations of follistatin. In this study, we investigated postprandial follistatin and activin A concentrations after intake of isocaloric amounts of protein, fat or glucose in obese subjects with and without previous RYGB to explore the regulation of follistatin by the individual macronutrients. Protein intake enhanced follistatin concentrations similarly in the two groups, while glucose and fat ingestion did not change postprandial follistatin concentrations. Concentrations of activin A were lower after protein intake compared with glucose intake in RYGB. Glucagon concentrations were also particularly enhanced by protein intake and tended to correlate with follistatin in RYGB. In conclusion, we demonstrated that protein intake, but not glucose or fat, is a strong stimulus for follistatin secretion in obese subjects and that this regulation is maintained after RYGB surgery.