The ABO blood group system is the most important factor in clinical transfusion medicine and is implicated in a number of human diseases. ABO antigens are not confined to red blood cells (RBCs) and are widely expressed in a variety of human cells and tissues. To date, many alleles with variant ABO expression have been identified and in many cases traced to one of the >250 reported genetic variations in the respective glycosyltransferase. The role of microRNAs (miRNAs) in the regulation of blood group antigens during erythropoiesis has not been addressed, however. Here, we show that miR-331-3p and miR-1908-5p directly target the mRNA of glycosyltransferases A and B. Expression levels of miR-331-3p and miR-1908-5p inversely correlated with levels of blood group A antigen. In addition, we found that overexpression of these miRNAs in hematopoietic stem cells led to a significantly reduced number of blood group A antigens per RBC. Simultaneous targeting of the transcription factor (TF) SP1 by miR-331-3p further enhanced these effects. The targeting rendered SP1 incapable of binding to the ABO gene promoter, causing further downregulation of blood group A antigen expression by up to 70%. Taken together, expression changes in these miRNAs may account for rare cases of weak A/B phenotypes that genetic variations in the glycosyltransferase coding region cannot explain. These results also suggest an explanation for the disappearance of ABH antigens during carcinogenesis and point to new therapeutic targets in ABO mismatched organ transplantation.Keywords: ABO blood group; HSC; SP1 transcription factor; glycosyltransferase; miRNA.