The lens epithelium-derived growth factor (LEDGF/p75) tethers the mixed-lineage leukemia (MLL1) protein complex to chromatin. Likewise, LEDGF/p75 tethers the HIV-1 pre-integration complex to chromatin. We previously demonstrated that expression of the C-terminal fragment fused to enhanced green fluorescent protein (eGFP) (eGFP-LEDGF(325-530)) impaired HIV-1 replication. Here, we explored this strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF(325-530) impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage-marker-depleted murine bone marrow cells. Expression of LEDGF(325-530) was associated with downregulation of the MLL target Hoxa9 and impaired cell cycle progression. Structure-function analysis revealed two small eGFP-fused LEDGF/p75 peptides, LEDGF(424-435) and LEDGF(375-386) phenocopying these effects. Both LEDGF(325-530) and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF(325-530) or LEDGF(375-386) fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9-induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction.