In spite of high complete remission rates in Acute Myeloid Leukemia (AML), little progress has been made in the long-term survival of relapsing AML patients, urging for the development of novel therapies. The CXCR4/SDF-1 axis is a potential therapeutic target in AML to reduce the enhanced survival and proliferation of leukemic cells, with current drug development efforts focusing on antagonists and blocking antibodies. The RNAi technology mediated by siRNA is a promising alternative; however, further development of clinically relevant siRNA carriers is needed since siRNA on its own is an incompetent silencing agent. Here, we report on lipid-substituted polymeric carriers for siRNA delivery to AML cells, specifically targeting CXCR4. Our results demonstrate an effective suppression of CXCR4 protein with the polymeric siRNA delivery in AML THP-1 cells. The suppression of CXCR4 as well as its ligand, SDF-1 (CXCL12), decreased THP-1 cell numbers due to reduced cell proliferation. The reduced proliferation was also observed in the presence of human bone marrow stromal cells (hBMSC), suggesting that our approach would be effective in the protective bone marrow microenvironment. The combination of CXCR4 silencing and cytarabine treatment resulted in more effective cytotoxicity when the cells were co-incubated with hBMSC. We observed a decrease in the toxicity of the lipopolymer/siRNA complexes when THP-1 cells were treated in the presence of hBMSC but this effect did not negatively affect CXCR4 silencing. In addition, siRNA delivery to mononuclear cells derived from AML patients led to significant CXCR4 silencing in 2 out of 5 samples, providing a proof-of-concept for clinical translation. We conclude that decreasing CXCR4 expression via lipopolymer/siRNA complexes is a promising option for AML therapy and could provide an effective alternative to current CXCR4 inhibition strategies.