Introduction: In Nigeria, there is paucity of data regarding indigenously established reference intervals for fasting plasma or serum insulin concentrations in the local populations. This study was designed to establish the reference interval of fasting plasma insulin (FPI) among apparently healthy young and middle-aged adults in a Nigerian population. The influence of age, gender, and anthropometric variables on FPI concentration were examined. Materials and Methods: The study was a cross-sectional descriptive study involving 210 reference individuals aged 18 to 64 years. Physical, anthropometric and biochemical variables were measured including FPI and fasting plasma glucose (FPG). The reference interval for FPI was determined using the non-parametric percentile method. Correlation studies between FPI and age, anthropometric indices, and HOMA-IR were carried out. Results: The study involved a total of 210 healthy normal-weight non-diabetic adults consisting of 110 males (52.4%) and 100 females (47.6%) as reference individuals. Reference intervals for FPI for the male, female, and total study participants were 0.1 –11.1mIU/L, 1.3 –13.2mIU/L and 0.1 – 13.03mIU/L respectively. There were statistically significant positive correlations between FPI and age (r = 0.001), WC (r = 0.0.302, p = 0.0001), WHR (r = 0.220, p = 0.0001), SBP (r = 0.137, p = 0.047) FPG (r = 0.165, p = 0.017) and HOMA-IR (r = 0.985, p = 0.0001). Conclusion: The reference interval of FPI using the Biointecho human insulin ELISA kit is 0.1–13.0mIU/L. This is not much at variance with 0.7–9.0mIU/L quoted by the kit manufacturer, but valued widely with that of other commercially available insulin assay kits from different manufacturers. Similar to reports of similar studies, FPI correlated positively with FPG, BMI. WC, BP and age.