The aim of this study is to determine the prevalence and incidence of vitamin B12 deficiency after esophagectomy for cancer. It is unknown if patients after esophagectomy with gastric tube reconstruction are at an increased risk for vitamin B12 deficiency. A cross-sectional cohort (group A) and a prospective cohort (group B) of patients who underwent esophagectomy for cancer in two tertiary referral centers in the Netherlands were included. Serum levels of holo-transcobalamin (Holo-TC) and methyl malonic acid (MMA) were determined. Vitamin B12 deficiency was defined as Holo-TC < 21 pmol/L and/or MMA > 0.45μmol/L. Vitamin B12 status was assessed in group A at a single time point between one and three years postoperatively and before and every three months after resection in group B. Ninety-nine patients were analyzed in group A. The median time between surgery and analysis of vitamin B12 deficiency was 19.3 months. In 11 of 99 (11%) patients, vitamin B12 deficiency was detected. In group B, 5 of 88 (5.6%) patients had vitamin B12 deficiency preoperatively, and another 9 (10.2%) patients developed vitamin B12 deficiency after the operation at a median time of 6 months postoperatively. The estimated one-year incidence of vitamin B12 deficiency was 18.2%. None of the patients with vitamin B12 deficiency had a megaloblastic anemia. Vitamin B12 deficiency can be anticipated in 18% of patients after esophagectomy with gastric tube reconstruction for cancer. During follow-up, Holo-TC and MMA levels should be measured to detect vitamin B12 deficiency and commence treatment timely.