WD repeat domain 5 (WDR5) is a prominent target for pharmacological inhibition in cancer through its scaffolding role with various oncogenic partners such as MLL and MYC. WDR5-related drug discovery efforts center on blocking these binding interfaces or degradation have been devoted to developing small-molecule inhibitors or degraders of WDR5 for cancer treatment. Nevertheless, the precise role of WDR5 in these cancer cells has not been well elucidated genetically. Here, by using an MLL-AF9 murine leukemia model, we found that genetically deletion of Wdr5 impairs cell growth and colony forming ability of MLL-AF9 leukemia cells in vitro or ex vivo and attenuates the leukemogenesis in vivo as well, which acts through direct regulation of ribosomal genes. Pharmacological inhibition of Wdr5 recapitulates genetic study results in the same model. In conclusion, our current study demonstrated the first genetic evidence for the indispensable role of Wdr5 in MLL-r leukemogenesis in vivo, which supports therapeutically targeting WDR5 in MLL-rearranged leukemia by strengthening its disease linkage genetically and deepening insights into its mechanism of action. Display omitted •Wdr5 conditional knockout mice were generated utilizing CRISPR/Cas9 KI approach.•Genetically loss of Wdr5 impairs MLL-r leukemogenesis in vitro and in vivo.•Wdr5 regulates MLL-r leukemia via cell-cycle, apoptosis, and cell differentiation.•Wdr5 functions through direct regulation of ribosomal genes.•Wdr5 inhibitor recapitulates the effect by loss of Wdr5 in MLL-r leukemia.