Context During the last two decades it became obvious that 3,5-diiodothyronine (3,5-T2), a well-known endogenous metabolite of the thyroid hormones thyroxine (T4) or triiodothyronine (T3), not only represents a simple degradation intermediate of the former but also exhibits specific metabolic activities. Administration of 3,5-T2 to hypothyroid rodents rapidly stimulated their basal metabolic rate, prevented high fat diet-induced obesity as well as steatosis and increased oxidation of long-chain fatty acids. Objective The aim of the present study was to analyze associations between circulating 3,5-T2 in human serum and different epidemiological parameters including age, sex or smoking as well as measures of anthropometry, glucose and lipid metabolism. Study Design and Methods 3,5-T2 concentrations were measured by a recently developed immunoassay in sera of 761 euthyroid participants of the population-based Study of Health in Pomerania (SHIP-TREND). Subsequently, analysis of variance and multivariate linear regression analysis were performed. Results Serum 3,5-T2 concentrations exhibited a right-skewed distribution, resulting in a median serum concentration of 0.24 nM (1st quartile: 0.20 nM; 3rd quartile: 0.37 nM). Significant associations between 3,5-T2 and serum fasting glucose, thyrotropin (TSH) as well as leptin concentrations were detected (p<0.05). Interestingly, the association to leptin concentrations seemed to be mediated by TSH. Age, sex, smoking and blood lipid profile parameters did not show significant associations with circulating 3,5-T2. Conclusion Our findings from a healthy euthyroid population may point towards a physiological link between circulating 3,5-T2 and glucose metabolism.