Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. It affects as many as 330 million individuals worldwide. This deficiency may determine neonatal jaundice, chronic nonspherocytic hemolytic anemia and acute hemolytic anemia induced by drugs, infections and broad bean ingestion. The efficacy of blood transfusion is decreased when the donor is G6PD deficient. In this study, we aimed at determining the prevalence of G6PD deficiency in blood donors of Mossoro, Brazil. Samples of 714 blood donors (576 men and 138 women; 343 white and 371 non-white) with ages ranging from 18 to 62 years and that accepted to participate in the study were analyzed. All participants answered a standard questionnaire. G6PD activity was analyzed by the methemoglobin reduction test with deficiency being confirmed by the semiquantitative test. The overall prevalence of G6PD deficiency in blood donors was 3.8%, similar to the rate described for others regions of Brazil. There was no significant statistical difference in the frequency of G6PD deficiency between men and women, nor between white and non-white blood donors. This relatively high frequency of G6PD deficiency highlights a need to screen blood donors for this condition.