AbstractKMT2C and KMT2Dare the most frequently mutated epigenetic genes in human cancers. WhileKMT2Cis identified as a tumor suppressor in acute myeloid leukemia (AML), the role ofKMT2Dremains unclear in this disease, though its loss promotes B cell lymphoma and various solid cancers. Here, it is reported thatKMT2Dis downregulated or mutated in AML and its deficiency, through shRNA knockdown or CRISPR/Cas9 editing, accelerates leukemogenesis in mice. Hematopoietic stem and progenitor cells and AML cells withKmt2dloss have significantly enhanced ribosome biogenesis and consistently, enlarged nucleolus, increased rRNA and protein synthesis rates. Mechanistically, it is found thatKMT2Ddeficiency leads to the activation of the mTOR pathway in both mouse and human AML cells.Kmt2ddirectly regulates the expression ofDdit4, a negative regulator of the mTOR pathway. Consistent with the abnormal ribosome biogenesis, it is shown that CX‐5461, an inhibitor of RNA polymerase I, significantly restrains the growth of AML withKmt2dloss in vivo and extends the survival of leukemic mice. These studies validateKMT2Das a de facto tumor suppressor in AML and reveal an unprecedented vulnerability to ribosome biogenesis inhibition.