SummaryDHX9 is a member of the DEAH (Asp-Glu-Ala-His) helicase family and regulates DNA replication and RNA processing. DHX9 dysfunction promotes tumorigenesis in several solid cancers. However, the role of DHX9 in MDS is still unknown. Here, we analyzed the expression ofDHX9and its clinical significance in 120 MDS patients and 42 non-MDS controls. Lentivirus-mediatedDHX9-knockdown experiments were performed to investigate its biological function. We also performed cell functional assays, gene microarray, and pharmacological intervention to investigate the mechanistic involvement of DHX9. We found that overexpression ofDHX9is frequent in MDS and associated with poor survival and high risk of acute myeloid leukemia (AML) transformation. DHX9 is essential for the maintenance of malignant proliferation of leukemia cells, and DHX9 suppression increases cell apoptosis and causes hypersensitivity to chemotherapeutic agents. Besides, knockdown ofDHX9inactivates the PI3K-AKT and ATR-Chk1 signaling, promotes R-loop accumulation, and R-loop-mediated DNA damage.