We conducted a clinical trial of low-dose decitabine plus aclacinomycin/cytarabine (AA) treatment (DAA) for 20 patients with refractory/relapsed de novo acute myeloid leukemia (AML) or AML transformed from myelodysplastic syndrome (MDS/AML) in order to examine its efficacy and tolerability. Additionally, P15(ink4b) methylation status was analyzed (for 15 patients) pre- and post-DAA treatment, and in vitro drug sensitivity tests were performed for seven patients (AA or AA + decitabine) to explore the role of decitabine in this combination treatment regimen. A total of 11 patients (55.0 %) achieved complete remission (CR) after DAA treatment, including 7 of whom reached CR after only one treatment course. The other two patients achieved partial remission. The median overall survival (OS) was 10 months for all 20 patients. The median OS for those who achieved CR was significantly longer than that of patients with no response (NR; P = 0.01). The treatment regimen was well tolerated, and there was no treatment-related mortality. The mean levels of P15(ink4b) methylation decreased significantly in six patients who achieved CR, whereas very few changes in P15 (ink4b) methylation were detected for the five patients with NR following DAA treatment. The data from the methyl thiazolyl tetrazolium assays showed that the inhibition rates of AA and DAA for tumor cells were identical. We conclude that induction therapy with DAA for refractory/relapsed de novo AML or MDS/AML achieved high levels of CR and improved OS and demonstrated adequate tolerance. Moreover, the decitabine component of DAA may function through a demethylation effect.