AMP deaminase (AMPD) catalyzes AMP to IMP and plays an important role in energy charge and nucleotide metabolism. Human AMPD3 deficiency is a type of erythrocyte-specific enzyme deficiency found in individuals without clinical symptoms, although an increased level of ATP in erythrocytes has been reported. To better understand the physiological and pathological roles of AMPD3 deficiency, we established a line of AMPD3-deficient [A3(−/−)] mice. No AMPD activity and a high level of ATP were observed in erythrocytes of these mice, similar to human RBC-AMPD3 deficiency, while other characteristics were unremarkable. Next, we created AMPD3 and pyruvate kinase (PK) double-deficient [PKA(−/−,−/−)] mice by mating A3(−/−) mice with CBA-Pk-1slc/Pk-1slc mice [PK(−/−)], a spontaneous PK-deficient strain showing hemolytic anemia. In PKA(−/−,−/−) mice, the level of ATP in red blood cells was increased 1.5 times as compared to PK(−/−) mice, although hemolytic anemia in those animals was not improved. In addition, we observed osmotic fragility of erythrocytes in A3(−/−) mice under fasting conditions. In contrast, the ATP level in erythrocytes was elevated in A3(−/−) mice as compared to the control. In conclusion, AMPD3 deficiency increases the level of ATP in erythrocytes, but does not improve anemia due to PK deficiency and leads to erythrocyte dysfunction.