Acute myeloid leukemia (AML) is the most common myeloid leukemia. It is highly malignant, thus, most patients with AML will relapse and die after traditional treatment. Stat3, a member of the signal transducer and activator of transcription (Stat) family, is involved in the development and progression of many tumors. The purpose of the study was to investigate whether the down-regulation of Stat3 expression by RNA interference is effective against human leukemia HL-60 cells. The results indicated that constitutively expressed Stat3 is present in human leukemia HL-60 cells, and the down-regulation of Stat3 expression caused significant induction of apoptosis as well as inhibition of proliferation in HL-60 cells. These data further demonstrated that Stat3 plays a critical role in human leukemia HL-60 cell apoptosis and proliferation. Thus, targeting Stat3 may be a useful adjunctive treatment strategy in AML.