Background/aim: The aim of this study is to determine the reference ranges of 25-hydroxyvitamin D3 (25(OH)vitD3) by an indirect method using data obtained from patients over the course of 3 years. Materials and methods: The 25(OH)vitD3 test results of the patients who applied to the medical biochemistry laboratory of Balıkesir State Hospital between the years of 2010 and 2014 were analyzed. Patient data were retrospectively taken from the laboratory information system. The 25(OH)vitD3 levels of patients were examined after exclusion of outliers. Patients were divided into subgroups according to season, sex, and age. The central 95% reference intervals were calculated using a nonparametric method. Results: Calculated reference intervals showed lower values than the recommended reference values of the manufacturer. In our study, 25(OH)vitD3 test results obtained for the reference values were 6.43-30.0 ng/mL for the percentile range of 2.5-97.5. For 25(OH)vitD3, the determined reference interval for our data was significantly different from the data provided by the manufacturer. Conclusion: This work should also be carried out in a healthy population. Data obtained from this study can be combined with the reference range determination studies for other regions in Turkey, and therefore it can contribute to the determination of the reference ranges of the Turkish community. This study is important for verification of the reference range recommended by the manufacturer.