An anti-CD44 monoclonal antibody, HI313, as a potential tool to treat Acute Myeloid Leukemia (AML) have been developed. The binding affinity of HI313 was measured by flow cytometry; suppression of NB4 cell line proliferation induced by HI313 was measured by MTS; effects of HI313 on cell cycle were assessed by flow cytometry; AML cell differentiation was assessed by detecting cell surface markers CD11b, CD14 and CD15. The results show that anti-CD44 antibody HI313 has relative high affinity to cells both high and low expression of CD44 and effectively suppressed the proliferation of NB4 cell line through blocking of cell cycle progress at G0/G1 phase. Furthermore, HI313 antibody can induce differentiation of cancer cells from AML patients. In conclusion, CD44 is a valid target for AML treatment and HI313 monoclonal antibody is a good candidate for therapeutic antibody development.