HLA-G5 is secreted by erythroblasts in all hematopoietic organs, suggesting a role for this protein in erythropoiesis. To examine this, we analyzed whether HLA-G5 affects the proliferation of UT7/EPO and HEL erythroleukemia cells and characterized the mechanism by which HLA-G5 influences erythropoietin receptor (EPOR) signaling. We show that HLA-G5 inhibits the proliferation of UT7/EPO cells, the EPOR signaling of which is similar to that of normal erythroid progenitors. HLA-G5-mediated inhibition was associated with reduced phosphorylation of JAK2 kinase and that of the downstream signaling proteins STAT-5 and STAT-3. Involvement of JAK2 in erythroid cell proliferation has been highlighted by the role of JAK2 V617F mutation in polycythemia vera (PV), a myeloproliferative disorder characterized by erythroid lineage overproduction. We demonstrate that HLA-G5 downregulates EPOR constitutive signaling of JAK2 V617F-expressing HEL cells, leading to inhibition of cell proliferation through G1 cell cycle arrest. Combination of HLA-G5 with JAK inhibitor I further decreases HEL cell growth. Clinical relevance is provided by analysis of PV patients who carry JAK2 V617F mutation, showing that HLA-G5 inhibits the formation of erythropoietin-independent erythroid colonies. Such HLA-G5-mediated inhibition constitutes a new parameter to be considered in the design of future approaches aimed at treating JAK2 V617F-positive myeloproliferative disorders.