This study was aimed to investigate the effects of methylation of runx3 gene promoter on pathogenesis of acute leukemia (AL) and its clinical significance. The methylation of runx3 gene promoter in cells of bone marrow or peripheral blood from 40 cases of AL and 10 healthy persons as well as in CHRF, U937 and K562 cell lines were assuaged by methylation specific polymerase chain reaction (MS-PCR), the expression of runx3 gene were detected with reverse transcription polymerase chain reaction (RT-PCR). The results indicated that no methylation was detected in all of cell lines and healthy persons while expression of runx3 gene could be deteted, methylation of runx3 gene promoter was found in 35% (14/40) AL patients and its percentage was significant higher than that healthy persons (0%), the difference in methylation for runx3 between two kinds of samples was statistically significant (p<0.05), while methylation rate in AML was 30.43% (7/23), ALL was 41.18% (7/17), there was no significant difference between them (p>0.25). All of methylated samples had no expression of runx3 gene. Patients without methylation of runx3 gene had a lower percentage of blasts in bone marrow and a higher complete remission rate of first chemotherapy than those with methylation of runx3 gene. It is concluded that methylation of runx3 gene promoter probably plays a role in the pathogenesis of AL and may have clinical significance in predicting prognosis of AL.