In Japan, most HbA1c measurements by enzymatic assays or immunoassays represent the HbA1c levels in the blood cell fraction obtained after centrifugation of the blood samples. The present study investigated that the blood cell enzymatic HbA1c assay (EA-HbA1c) was compared with whole blood HbA1c in patients with iron deficiency anemia (IDA). Study 1: EA-HbA1c levels using blood cell samples (blood cell EA-HbA1c) and high-performance liquid chromatography (HPLC)-HbA1c levels using whole blood samples (whole blood HPLC-HbA1c) were measured in 15 IDA patients with Hb < 8 g/dL and transferrin saturation (TSAT) < 20%, and the correlations between the blood cell EA-HbA1c/whole blood HPLC-HbA1c ratio (%) and various IDA indicators [mean corpuscular hemoglobin concentration (MCHC), TSAT, and logarithmically transformed ferritin (log-FER)] were examined. Study 2: Blood cell EA-HbA1c levels were compared with EA-HbA1c levels using whole blood samples (whole blood EA-HbA1c) and whole blood HPLC-HbA1c levels in 10 of the above IDA patients. In Study 1, blood cell EA-HbA1c levels were significantly higher than whole blood HPLC-HbA1c levels, and the blood cell EA-HbA1c/whole blood HPLC-HbA1c ratio (%) showed significant negative correlations with MCHC, TSAT, and log-FER in the IDA patients. In Study 2, blood cell EA-HbA1c levels were significantly higher than whole blood EA-HbA1c and whole blood HPLC-HbA1c levels in IDA patients. The present study showed for the first time that blood cell EA-HbA1c are higher than whole blood HbA1c in IDA patients. In IDA patients with marked anemia, EA-HbA1c levels using blood cells show different results from those using whole blood.