INTRODUCTION Gallstone disease is associated with insulin resistance, type 2 diabetes mellitus, and increased risk of incident ischemic heart disease. It is known that the profile of branched‑chain amino acids (BCAAs) is altered in cardiac diseases as well as metabolic diseases, such as diabetes and obesity. The role of BCAAs in gallstone disease is still not known. OBJECTIVES The aim of this study was to evaluate the concentration of essential amino acids and incretin hormones in patients with cholecystolithiasis. PATIENTS AND METHODS The study included 31 patients with cholecystolithiasis and 25 gallstone‑free controls. The levels of free exogenous and endogenous amino acids, bile acids, glucagon‑like peptide 1, glucose‑dependent insulinotropic polypeptide, ghrelin, C‐peptide, and insulin were measured in the fasting state and 1 hour after consumption of a 300‑kcal mixed meal. RESULTS The mean fasting and postprandial levels of valine, isoleucine, leucine, and lysine were higher in the study group than in controls (all P <0.01). The percentage increase in concentrations of amino acids after a meal were similar in both groups of patients. The mean fasting concentrations of C‑peptide and glucagon‑like peptide 1 were higher in the study group than in the control group (P = 0.004 and P = 0.03, respectively), and the median postprandial concentration of C‑peptide was higher in the study group as compared with the control group (P = 0.03). CONCLUSIONS Diabetes mellitus, coronary heart disease, and gallstone disease might have common genetic and environmental antecedents. However, higher plasma levels of BCAAs observed in patients with gallstone disease may be partly responsible for metabolic complications observed in these patients.