Background: Elderly people have increased risk factors for low serum vitamin D levels, which is worsened among the black race. Therefore, elderly Africans constitute a reference population for vitamin D study. Aim: The aim of this study was to establish the reference interval of serum 25-hydroxyvitamin D (25(OH)D) among an African elderly population. Methodology: This was a cross-sectional study of rural community dwellers in Enugu, south-eastern Nigeria aged 50 years and above, that satisfied the criteria of the reference population. Ethical approval and informed consent were obtained. Venous blood was collected from reference individuals and serum 25(OH)D was determined by enzyme-linked immunosorbent assay.Data were analysed using a non-parametric, bootstrap method to obtain the central 95% reference limits and 90% confidence intervals of the lower and upper limits of the reference interval respectively. Results: One hundred and twenty-four (62 males and 62 females) participants were recruited. The median (25th -75th percentile) of serum 25(OH)D was 56 (35 – 71) ng/ml. The 2.5th percentile defined the lower reference limit and it was 21 ng/ml with 90% confidence interval (20 – 23) ng/ml; while the 97.5th percentile defined the upper reference limit and it was 93 ng/ml with 90% confidence interval (90 – 98) ng/ml. Conclusion: The reference interval for serum 25(OH)D for the selected African elderly population in Enugu, Nigeria was determined to be 21 to 93 ng/ml. Keywords: Bootstrap method; elderly; reference interval; vitamin D.