Recent studies suggest that FK506 binding protein 51 (FKBP51), a negative regulator of glucocorticoid response, encoded by FKBP5, may influence insulin action. The aim of the present study was to assess the relationship between subcutaneous adipose tissue (AT) and skeletal muscle FKBP5 expression in relation to insulin sensitivity in healthy individuals and to study its regulation by insulin and circulating free fatty acid (FFA) elevation.The study group comprised 96 male subjects, 49 normal-weight and 47 overweight/obese. Hyperinsulinemic clamp, subcutaneous AT and skeletal muscle biopsies were performed. In a subgroup of 20 subjects, two 6 h clamps were performed, with and without Intralipid/heparin infusion, and tissue biopsies were obtained before and after each clamp.AT FKBP5 expression was lower in overweight/obese individuals in comparison with normal-weight individuals (p = 0.004). Muscle FKBP5 expression did not differ between the groups, however, it was inversely related to insulin sensitivity (r = -0.32, p = 0.002). FKBP5 expression decreased in AT (p = 0.003) and increased in muscle (p < 0.0001) after insulin infusion. Intralipid/heparin diminished insulin-induced increase in muscle FKBP5.Our data show that lower AT FKBP5 expression is related to obesity, whereas muscle FKBP5 expression is associated with insulin resistance. AT and muscle FKBP5 expression is differentially regulated by insulin.