The oncoprotein RUNX1-ETO is the fusion product of t(8;21)(q22;q22) and constitutes one of the most common genetic alterations in acute myeloid leukemia (AML). Abnormal c-KIT overexpression is considered an independent negative prognostic factor for relapse and survival in t(8;21) AML patients. However, the molecular mechanism of high c-KIT expression in t(8;21) AML remains unknown. In this study, we detected RUNX1-ETO and c-KIT gene expression in AML-M2 patients and verified the overexpression of c-KIT in t(8;21) AML patients. We also found that c-KIT overexpression was a poor prognostic indicator in RUNX1-ETO positive AML patients, but not in RUNX1-ETO negative AML patients. We used the dual-luciferase and ChIP assays to demonstrate that the RUNX1-ETO protein epigenetically trans-activates c-KIT by binding to the c-KIT promoter and recruiting the histone acetyltransferase P300 to the c-KIT promoter, elucidating the mechanism of the abnormally increased c-KIT expression in t(8;21) AML patients. Moreover, pharmacological studies revealed that C646, a P300 inhibitor, could inhibit proliferation, induce apoptosis and arrest the cell cycle more effectively in RUNX1-ETO positive cells than in negative ones. The levels of c-KIT and RUNX1-ETO proteins were also decreased with C646 treatment in RUNX1-ETO positive cells. These findings suggested that P300 could be a therapeutic target and that C646 could be used as a potential treatment for RUNX1-ETO positive AML patients. Interestingly, using the dual-luciferase assay, we also found that the binding capacity of RUNX1-ETO9a, a truncated RUNX1-ETO isoform, to the c-KIT promoter was stronger than that of RUNX1-ETO, suggesting RUNX1-ETO9a as another valuable therapeutic target in t(8;21) AML.