Aspergillus flavus and its toxic aflatoxins secondary metabolites contaminate food and grains, posing a severe threat to human health and leading to liver cancer. Here, we demonstrated that cycloleucine blocked aflatoxin B1 synthesis by inhibiting N6-methyladenosine (m6A) methylation modification of messenger RNA (mRNA). m6A Methylation Immunoprecipitation Sequencing (m6A MeRIP-Seq)-based comprehensive transcriptome-wide m6A profiling identified 102 differentially expressed genes that underwent m6A modification, of which 22 hypermethylated genes were downregulated and 49 hypomethylated genes were upregulated, suggesting a negative correlation between m6A methylation and gene expression. Notably, cycloleucine inhibited aflatoxin B1 production via multiple targets. The m6A sites of several key genes involved in the aflatoxin B1 biosynthesis pathway were significantly enriched in the coding sequence and around the stop codon, resulting in their downregulation. Furthermore, m6A methylation on genes related to the aflatoxin B1 biosynthesis pathway led to reduced mRNA stability. Cycloleucine inhibition of aflatoxin B1 production highlights its potential as an agent for removing mycotoxins in environmental pollution. Environmental implicationAflatoxins, highly carcinogenic secondary metabolites produced by Aspergillus flavus, frequently contaminate crops such as peanut, corn, wheat and sesame leading to irreversible loss in the quality and yield of agricultural products and posing serious threats to food safety. Aflatoxins has also been linked to developmental delays and liver cancer in humans. In our study, ‘monitoring aflatoxin concentrations and its bioaccumulation in organisms’ has been conducted. The results demonstrated that aflatoxin production in A. flavus was completely blocked after cycloleucine treatment. Additionally, we demonstrated that inhibition of aflatoxin was linked to N6-methyladenosine methylation of multiple genes in aflatoxin biosynthesis pathway.