AML1-ETO fusion protein (AE) is generated by t(8;21)(q22;q22) chromosomal translocation, which is one of the most frequently observed structural abnormalities in acute myeloid leukemia (AML) and displays a pivotal role in leukemogenesis. The histone acetyltransferase p300 promotes self-renewal of leukemia cells by acetylating AE and facilitating its downstream gene expression as a transcriptional coactivator, suggesting that p300 may be a potential therapeutic target for AE-positive AML. However, the effects of p300 inhibitors on leukemia cells and the underlying mechanisms have not been extensively investigated. In the current study, we analyzed the anti-leukemia effects of C646, a selective and competitive p300 inhibitor, on AML cells. Results showed that C646 inhibited cellular proliferation, reduced colony formation, evoked partial cell cycle arrest in G1 phase, and induced apoptosis in AE-positive AML cell lines and primary blasts isolated from leukemic mice and AML patients. Nevertheless, no significant inhibitory effects were observed in granulocyte colony-stimulating factor-mobilized normal peripheral blood stem cells. Notably, AE-positive AML cells were more sensitive to lower C646 doses than AE-negative ones. And C646-induced growth inhibition on AE-positive AML cells was associated with reduced global histone H3 acetylation and declined c-kit and bcl-2 levels. Therefore, C646 may be a potential candidate for treating AE-positive AML.