Purpose: Hemoglobin A1c (A1C) is an invaluable indicator of glycemic control, and, since 2010, the A1C test has been recommended as one of the diagnostic criteria for diabetes. There are some factors that interfere with the measurement of A1C. If clinicians are not aware of these limitations, they may be misled in their diagnosis or manage diabetes inappropriately. This study explored the factors associated with A1C values≦4% and identified the causes of falsely low A1C values. Methods: Between January 2010 and December 2011, we used an ion-exchange high-performance liquid chromatogram (HPLC) to measure A1C in our hospital. All samples with A1C values≦4% were submitted for hemoglobin electrophoresis, complete blood count (CBC) and glucose-6-phosphate dehydrogenase (G6PD) levels. Medical records were reviewed to identify conditions that might be associated with these low A1C values. Results: Of the 64,653 samples analyzed, 27 had A1C values≦4%. The interfering factors could be divided into four categories: 1) eight cases with hemoglobin variant J (Hb J); 2) nine with hemolytic anemia, including 4 with G6PD deficiency and 4 taking ribavirin for hepatitis C; 3) seven with upper gastrointestinal bleeding, including 5 with cirrhosis of the liver; 4) 3 with anemia of undetermined cause. The Hb variant group had lower A1C values (p=0.008) and the non-variant group had lower hemoglobin levels (p=0.002). Conclusions: Hemoglobin variant J, acute blood loss and hemolytic anemia were the major causes of falsely low A1C when the ion-exchange HPLC method of testing was used. G6PD deficiency and anti-viral drug ribavirin were the two most common causes of hemolysis. In addition to visual inspection of the chromatogram to detect abnormalities, CBC and G6PD should be determined in order to identify the reasons for falsely low A1C values.