The study aimed at elucidation the bio-function of ash1 on epigenetic regulation in A. flavus. The ash1 gene deletion strain (Δash1) and ash1 Complementation strain (Com-ash1) were constructed by homologous recombination. It was found in morphology analysis that conidial production in the Δash1 strain was increased compared other two strains. The expression level of conidia development regulator abaA and brlA genes were further detected by Q-PCR, the result showed ash1 gene depressed the expression of abaA gene at 48h, and the expression of brlA gene at 48 and 72h significantly in WT and Com-ash1 strain compared with Δash1 strain. Ash1 gene was found involved in mycelia and sclerotia development. On YPD medium, the flavus colonies were significantly smaller than that of WT and com-ash1 strains when ash1 gene was deleted. It showed ash1 gene deletion significantly affect the development of sclerotia in 6 d culture, none sclerotia was found formed in Δash1 strain compare to the other two flavus strains. The expression level of sclerotia regulators (nsdC and nsdD) were monitored with Q-PCR. The results showed ash1 gene significantly up-regulate nsdC at 48 and 72 h, and up-regulate nsdD at 72 h. It was found in secondary metabolites bio-synthesis analysis that Aflatoxin B1 (AFB1) expression level was dramatically down-regulated by TLC analysis when ash1 gene was deleted. The TLC result was further confirmed with HPLC. The expression level of AFB1 bio-synthesis regulatorsand enzymesat 48 and 72 h were further analyzed with Q-PCR. The results revealed that ash1 gene down-regulated the upstream regulator (aflR) and enzymes (aflCand aflD) in AFB1 synthesis pathway, and up-regulated the downstream regulator (aflS) and enzymes (aflK and aflQ). The secondary metabolites from WT and Δash1 strains were further analyzed by HPLC with ultraviolet full wavelength scanning. It was found that a significant peak near 15 min disappear from Δash1 strains compared to WT strain at 254 nm, and the chemical characteristics of the peak would be further analysis by LC-MS/MS. On peanut surface, the AFB1 bio-synthesis and the conidia producing capacity was significantly down-regulated when ash1 gene was deleted, which suggested ash1 gene is required in the process of mycotoxin contamination and offspring transmission of A. flavus among crops. The study showed that Epigenetic regulation-histone lysine methylation transferase Ash1 play an critical role in morphogenesis, secondary metabolites bio-synthesis, and pathogenicity of A. flavus. And more attention should be paid on the role of histone methylation transferases on fungal development, secondary metabolism, and even pathogenicity.