microRNA-34a (miRNA-34a) plays an important role in the pathogenesis of leukemia. This study aimed to explore its role in the proliferation of HL-60 cells and the correlation with some of its predicted target genes: the cyclin-dependent kinase 4 (CDK4), the oncogene MYB and the silent information regulator 1 (SIRT1). We first analyzed the expression of miR-34a, CDK4, MYB and SIRT1 in peripheral blood samples from acute leukemia (AL) patients and healthy controls, and conducted a correlation analysis. HL-60 cells were then transfected with miR-34a and control 'scramble' miRNA, and quantitative RT-PCR and western blotting were used to analyze the effects of the interfering sequence in HL-60 cells. The expression of miR-34a was significantly reduced in AL patients compared to healthy controls (P<0.01), and negatively correlated with the expression of CDK4 and MYB. Sub-group analysis revealed that the expression of MYB was significantly lower in AL children <3 years old compared to those >3 years. Following the transfection of HL-60 cells with miR-34a, the mRNA level of CDK4, MYB and SIRT1 decreased by 53.2, 43.3 and 33.5%, respectively, compared to the control, similarly to the respective changes in protein levels. This study showed that the expression of miR-34a negatively correlates with the expression of CDK4 and MYB in pediatric patients with acute leukemia. miRNA-34a downregulates the expression of the CDK4, MYB and SIRT1 genes in vitro; it may thus represent a novel therapeutic target for acute leukemia.