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Transitioning from total to active vitamin B12 testing may influence diagnostic categorisation and costs. In this retrospective population study, a total of 181 794 vitamin B12 results from primary care requests in a UK laboratory were analysed -90 036 using total B12 assay before transitioning, and 91 758 using active B12 assay after transitioning- according to the National Institute for Health and Care Excellence (NICE)-defined thresholds. Compared with total B12, active B12 testing identified proportionately fewer patients as deficient (2.1% vs 6.5%) or indeterminate (37.0% vs 46.2%), and more as deficiency-unlikely (60.9% vs 47.3%). Although active B12 improved classification and may reduce the need for confirmatory testing by classifying fewer individuals as indeterminate, it incurred substantially higher cost, with an estimated 26-fold increase in expenditure to identify one deficient patient based on unit test cost in the NICE guidance. While active B12 may enhance diagnostic precision, its financial impact could limit broader adoption for routine testing unless test costs decrease.