DHX9 is a multifunctional DExH-box RNA helicase with important roles in the regulation of transcription, translation, and maintenance of genome stability. Elevated expression of DHX9 is evident in multiple cancer types, including colorectal cancer. Microsatellite instable-high (MSI-H) tumors with deficient mismatch repair (dMMR) display a strong dependence on DHX9, making this helicase an attractive target for oncology drug discovery. In this report, we show that DHX9 knockdown increased RNA/DNA secondary structures and replication stress, resulting in cell-cycle arrest and the onset of apoptosis in cancer cells with MSI-H/dMMR. ATX968 was identified as a potent and selective inhibitor of DHX9 helicase activity. Chemical inhibition of DHX9 enzymatic activity elicited similar selective effects on cell proliferation as seen with genetic knockdown. In addition, ATX968 induced robust and durable responses in an MSI-H/dMMR xenograft model but not in a microsatellite stable/proficient MMR model. These preclinical data validate DHX9 as a target for the treatment of patients with MSI-H/dMMR. Additionally, this potent and selective inhibitor of DHX9 provides a valuable tool with which to further explore the effects of inhibition of DHX9 enzymatic activity on the proliferation of cancer cells in vitro and in vivo. Significance: DHX9 is required in cancer cells with deficient mismatch repair and can be inhibited by ATX968, providing a promising strategy for the development of precision cancer therapeutics.