Objective To investigate the consistency of 25-hydroxyvitamin D (25-OH-VD) test results before and after a reagent generation change, assess the risk of misclassification associated with continued use of the previous reference interval after reagent replacement, and provide experimental evidence for the verification and revision of reference intervals following reagent changes in clinical laboratories. Methods The comparability of the two reagent generations was evaluated by simultaneously measuring samples using the pre-upgrade reagent (25-OH-VD Ⅰ) and the post-upgrade reagent (25-OH-VD Ⅲ). Healthy individuals who underwent physical examinations at the Health Management Center of West China Hospital, Sichuan University between March and June 2024 (25-OH-VD Ⅰ group, n=8203) and between March and June 2025 (25-OH-VD Ⅲ group, n=8203) were included. The distributions of results obtained using the two reagent generations across different concentration categories were compared. The consistency of the results was further evaluated through stratified analyses according to parathyroid hormone concentrations, and differences in the reference intervals between the two reagent generations were retrospectively analyzed. Results The relative differences between the results obtained using the 25-OH-VD Ⅲ and 25-OH-VD Ⅰ reagents ranged from –17% to +6%. In the 25-OH-VD deficiency category (<30 nmol/L), 428 individuals were identified using the 25-OH-VD Ⅰ reagent and 1195 using the 25-OH-VD Ⅲ reagent. In the insufficiency category (30-50 nmol/L), the corresponding numbers were 3324 and 4171, respectively. In the three parathyroid hormone strata of <1.6, 1.6-6.9, and >6.9 pmol/L, the median (lower quartile, upper quartile) of the 25-OH-VD concentrations measured using the 25-OH-VD Ⅲ reagent were 55.0 (41.9, 63.2), 44.9 (35.4, 57.6), and 39.1 (31.3, 49.4) nmol/L, respectively, all of which were lower than the corresponding values obtained using the 25-OH-VD Ⅰ reagent [70.8 (63.9, 79.1), 53.8 (42.6, 68.2), and 46.0 (37.1, 59.1) nmol/L, respectively; all P<0.05]. The P2.5-P97.5 reference interval for the 25-OH-VD Ⅲ reagent was 23.0-96.0 nmol/L, compared with 27.6-110.6 nmol/L for the 25-OH-VD Ⅰ reagent, representing decreases of 4.6 nmol/L in the lower limit and 14.6 nmol/L in the upper limit. Conclusions Significant differences were observed between the 25-OH-VD Ⅲ and 25-OH-VD Ⅰ reagents. Direct application of the reference interval established for the previous reagent may lead to overdiagnosis of vitamin D deficiency. Following a reagent generation change, laboratories should communicate the resulting differences to clinicians and, when necessary, establish a correction equation or revise the laboratory-specific reference interval.