Abstract We investigated the role of PRMT5 in myeloproliferative neoplasm (MPN) pathogenesis and aimed to elucidate key PRMT5 targets contributing to MPN maintenance. PRMT5 is overexpressed in primary MPN cells, and PRMT5 inhibition potently reduced MPN cell proliferation ex vivo. PRMT5 inhibition was efficacious at reversing elevated hematocrit, leukocytosis, and splenomegaly in a model of JAK2V617F+ polycythemia vera and leukocyte and platelet counts, hepatosplenomegaly, and fibrosis in the MPLW515L model of myelofibrosis. Dual targeting of JAK and PRMT5 was superior to JAK or PRMT5 inhibitor monotherapy, further decreasing elevated counts and extramedullary hematopoiesis in vivo. PRMT5 inhibition reduced expression of E2F targets and altered the methylation status of E2F1 leading to attenuated DNA damage repair, cell-cycle arrest, and increased apoptosis. Our data link PRMT5 to E2F1 regulatory function and MPN cell survival and provide a strong mechanistic rationale for clinical trials of PRMT5 inhibitors in MPN. Significance: Expression of PRMT5 and E2F targets is increased in JAK2V617F+ MPN. Pharmacologic inhibition of PRMT5 alters the methylation status of E2F1 and shows efficacy in JAK2V617F/MPLW515L MPN models and primary samples. PRMT5 represents a potential novel therapeutic target for MPN, which is now being clinically evaluated. This article is highlighted in the In This Issue feature, p. 1611