Thrombocytopenia is a common, often fatal complication experienced by patients with myelodysplastic syndromes (MDS). 5-aza-2′-deoxycytidine (decitabine) has been used to treat MDS patients with thrombocytopenia with a response rate of 45–50%. However, the mechanism of its effects on megakaryocytes remains unclear. In the present study, the effect of decitabine on megakaryocyte maturation was investigated. A total of 20 MDS patients diagnosed with thrombocytopenia were enrolled, including 16 refractory anemia with excess blasts (RAEB)-1 patients and 4 RAEB-2 patients], in addition to 20 leukemia patients that had achieved complete remission and 20 healthy donors. Overall, 65% of MDS patients exhibited a response to decitabine, with an increase in platelet count identified in 80% of patients. In the MDS group, the mean platelet count was significantly increased following one cycle of decitabine chemotherapy (36.85±24.54 vs. 84.90±61; P=0.001); however, no significant difference in megakaryocyte number was identified prior to and following treatment. Additionally, bone marrow mononuclear cells of the MDS patients were cultured in vitro with various concentrations of decitabine (0.0, 2.0, 2.5, 3.0 µM), and cluster of differentiation (CD)41 levels were examined via flow cytometry. The MDS and normal control groups exhibited the highest levels of CD41 expression following treatment with 2.0 µM decitabine (mean fluorescence intensity, 294.07±47.34 and 258.95±28.05, respectively). In conclusion, these results indicate that the DNA-hypomethylating agent, decitabine, may induce the differentiation and maturation of myelodysplastic megakaryocytes in MDS patients, even at low concentrations. Thus, the repeated administration of decitabine at lower doses in MDS patients may be useful in clinical practice, and may lead to the development of alternative treatments for other diseases of abnormal megakaryocyte differentiation, such as idiopathic thrombocytopenic purpura, however, future studies are required to investigate this.