Genomic DNA demethylation may activate silenced gene,which alters secondary metabolite spectrum,and therefore it is a completely novel approach for strain improvement. Using different concentrations of DNA methyltransferase inhibitor 5-azacytidine(5-azaC)to treat the Cordyceps militaris CM-L1,then the genomic DNA methylation level reduced. Further,the improved strains LB-C3 and LD-A7 were acquired by screening with high performance liquid chromatography,and in which the contents of cordycepin in mycelium increased by 127% and 144.3% respectively. LD-A7 could not form a fruiting body,which was speculated to be related to the role of DNA methylation changed. The stability was investigated after 5 times subculture using the content of cordycepin as an indicator. With the subculture times increased,the methylated DNA in the genome increased,and the cordycepin in mycelium of liquid fermentation significantly decreased;however,the content of cordycepin in fruiting bodies was very stable,indicating that improved strains were better suited to the cultivation rather than production in industrial fermentation.