Recently, a central role of butyrylcholinesterase (BChE) in pathogenesis of obesity has been proposed. As BChE was shown to inactivate a hunger hormone, ghrelin, it has been suggested to control appetite and glucose metabolism by regulating ghrelin signaling in hypothalamus. The aim of our study was therefore to investigate the relationship between obesity induced by high-fat/high-fructose diet and BChE activity. We studied impact of the diet-induced obesity on serum BChE and vise-versa an effect of BChE inhibition on obesity development. Adults male Wistar rats were fed with standard or high-fat/high-fructose diet, with or without parallel treatment with BChE inhibitor for 16 weeks. Food consumption, water intake and body weight were followed regularly. At the end of the experiment, the animals were sacrificed and effect of the diet and treatment was studied in adipose tissues and selected organs. BChE activity was determined in serum by Ellman‘s assay and serum levels of total and active ghrelin, leptin, and insulin were assessed by ELISA method. Rats fed with high-fat/high-fructose diet had higher caloric intake despite lower food income which was accompanied with higher body and adipose tissue weights and fat accumulation in tissues. Serum levels of leptin were increased while other tested hormones mediating energy homeostasis were unaffected. Importantly, serum BChE activity was increased. Systemic BChE inhibition had no impact on any of the studied parameters. Our results confirm relationship between serum BChE activity and high-fat/high-fructose-induced obesity, but do not support hypothesis that BChE regulates centrally ghrelin-controlled appetite. Supported by APVV-22-0541.