Previous studies have suggested that iron-deficiency anemia affects glycosylated hemoglobin (HbA1c) measurements, but the results were contradictory. We conducted a retrospective case-control study to determine the effects of iron deficiency on HbA1c levels. Starting with the large computerized database of the Italian Hospital of Desio, including data from 2000 to 2016, all non-pregnant individuals older than 12 years of age with at least one measurement of HbA1c, cell blood count, ferritin, and fasting blood glucose on the same date of blood collection were enrolled. A total of 2,831 patients met the study criteria. Eighty-six individuals were diagnosed with iron-deficiency anemia, while 2,745 had a normal iron state. The adjusted means of HbA1c were significantly higher in anemic subjects (5.59% [37.37 mmol/mol]), than those measured in individuals without anemia (5.34% [34.81 mmol/mol]) (<0.0001). These results suggest that clinicians should be cautious about diagnosing prediabetes and diabetes in individuals with anemia.