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The present study aimed to explore the function of miR‑34b promoter methylation in cell proliferation in children's acute leukemia. Quantitative PCR and methylation‑specific PCR were performed to measure the levels of miR‑34b and its promoter methylation in normal cells, eight leukemia cell lines as well as primary leukemic cells isolated from patients newly diagnosed with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and mixed lymphocytic lymphoma. miR‑34b levels in leukemia cell lines and primary leukemic cells were significantly lower than those in normal cells. The miR‑34b promoter was found to be methylated in all leukemia cell lines, 24 of 31 ALL patients and 8 of 19 AML patients, but not in the 23 normal controls. miR‑34b expression and methylation of its promoter were not associated with most clinical parameters assessed; however, miR‑34b levels in prednisone‑sensitive ALL were significantly different from those in insensitive ALL. A cell counting kit‑8 assay showed that transfection of miR‑34b mimics into K562 cells inhibited their proliferation. Furthermore, treatment with the demethylating agent 5‑aza‑2‑deoxycytidine significantly enhanced miR‑34b expression levels and decreased the methylation status of its promoter in HL‑60 and K562 cells. In conclusion, the results of the present study indicated that in pediatric leukemia cells and leukemia cell lines, the expression of miR‑34b is inhibited by methylation of its promoter, which impairs the restraining effects of miR‑34b on cell proliferation. It was also indicated that the expression of miR‑34b in ALL patients may affect their response to early treatments.