<p>MicroRNAs (miRNAs) are a class of non-coding RNAs involved in a variety of pathophysiological processes. We have previously reported that the abnormally high expression of miR-99a is associated with drug resistance and poor prognosis in acute myeloid leukemia. However, the impact of miR-99a on normal hematopoiesis is not well understood. To investigate the effect of aberrant miR-99a overexpression on hematopoietic stem and progenitor cells (HSPCs), we overexpressed miR-99a in human umbilical cord blood CD34⁺ cells. We observed that miR-99a overexpression increased the proliferation, self-renewal capacity, and transplantation efficiency of HSPCs with or without a clonal hematopoiesis-associated mutation (<i>JAK2</i>^V617F). Meanwhile, we found that overexpression of miR-99a blocked the maturation and differentiation of granulocytes/monocytes and erythrocytes. We then identified <i>NIPBL</i> as a direct target of miR-99a. <i>NIPBL</i> knockdown in HSPCs showed a phenotype similar to miR-99a overexpression. In this study, we elucidate that abnormally high expression of miR-99a can enhance the proliferative capacity of HSPCs but inhibit myeloid differentiation and maturation. Taken together, our work has uncovered important roles for miR-99a in regulating HSPCs by enhancing the proliferation and self-renewal capacity of HSPCs but inhibiting differentiation, which play important roles in leukemic transformation.</p>