Background: Despite the increasing interest towards the biological role of L-ergothioneine, little is known about the serum concentrations of this unusual aminothiol in older adults. We addressed this issue in a representative sample of community-dwelling middle-aged and older adults. Methods: Body mass index, estimated glomerular filtration rate, serum concentrations of L-ergothioneine, taurine, homocysteine, cysteine, glutathione, cysteinylglycine, and glutamylcysteine were evaluated in 439 subjects (age 55-85 years) randomly selected from the Hunter Community Study. Results: Median L-ergothioneine concentration in the entire cohort was 1.01 IQR 0.78-1.33 μmol/L. Concentrations were not affected by gender (P = 0.41) or by presence of chronic medical conditions (P = 0.15). By considering only healthy subjects, we defined a reference interval for L-ergothioneine serum concentrations from 0.36 (90% CI 0.31-0.44) to 3.08 (90% CI 2.45-3.76) μmol/L. Using stepwise multiple linear regression analysis L-ergothioneine was negatively correlated with age (rpartial = 20.15; P = 0.0018) and with glutamylcysteine concentrations (rpartial = 20.13; P = 0.0063). Conclusions: A thorough analysis of serum L-ergothioneine concentrations was performed in a large group of community-dwelling middle-aged and older adults. Reference intervals were established. Age and glutamylcysteine were independently negatively associated with L-ergothioneine serum concentration.