Transcobalamin-bound vitamin B12, or holotranscobalamin (HoloTC), a biologically active form, is believed to be a sensitive marker of vitamin B12 deficiency. We investigated the prevalence of vitamin B12 deficiency in gastrectomized patients using HoloTC and total vitamin B12 to determine the diagnostic utility of HoloTC. We enrolled 128 gastrectomized patients and measured serum HoloTC, total vitamin B12, homocysteine (Hcy), and complete blood count (CBC). HoloTC values were also obtained from 100 healthy controls. The precision of HoloTC measurement was good and the normal range of HoloTC was set at ≥42.48 pmol/L. Among the 128 gastrectomized subjects, HoloTC was low (<42.48 pmol/L) in 32 patients (25.0%) and total vitamin B12 was low (<189 pg/ml) in 10 patients (7.8%). Among the patients who had total vitamin B12 concentrations in the borderline range (189-400 pg/ml), 44% had low HoloTC concentrations. Clinical findings as well as Hcy concentrations suggested vitamin B12 deficiency in this subset of patients. The patients with both low total vitamin B12 and low HoloTC had significantly higher Hcy concentrations than those with either normal total vitamin B12 or normal HoloTC. Although a quarter of gastrectomized patients had low HoloTC, some of these had normal total vitamin B12 concentrations. Our study suggests that serum HoloTC is a more sensitive marker than total vitamin B12 in diagnosing vitamin B12 deficiency.