Increased insulin concentration is a surrogate for insulin resistance and early assessment of fasting insulin may help in identifying those who are potentially at high risk of type 2 diabetes, hypertension, and cardiovascular disease. The aim of this study was to determine age- and sex-related reference values for serum insulin and insulin resistance/sensitivity indices in Iranian subjects.Serum insulin levels were measured by electrochemiluminescence immunoassay in 5786 participants of the Tehran Lipid and Glucose Study. After application of exclusion criteria, 309 non-obese healthy subjects (124 men and 185 women), aged 24–83y, were included. The International Federation of Clinical Chemistry guidelines (non-parametric method) and the robust method were used for determining reference values.Overall 95% reference values for fasting insulin were 1.61–11.37, 2.34–11.98, and 2.11–12.49μU/mL in men, women, and total population respectively. Mean fasting insulin concentration showed a decreasing trend with age in both genders (p for trend ≤0.001). Age, waist circumference, and systolic blood pressures were biological determinants of fasting insulin in both genders; in addition, insulin was modulated by triglycerides in men and fasting glucose in women. Reference intervals for HOMA1-IR, HOMA2-IR, and QUICKI were 0.63–2.68, 0.40–1.80, and 0.33–0.42, respectively.This study presents the first set of reference values for fasting serum insulin to be 2–12μU/mL for both genders in a healthy sample of Iranian adults along with the reference values for insulin resistance/sensitivity indices. These values could be used for identifying subjects with insulin resistance in epidemiological and clinical research.