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The compulsive and insidious secondary metabolite aflatoxin B1, produced by the opportunistic fungi Aspergillus flavus, upholds a distinguished place in midst of the toxicants causing fatal hazards to humans. Aflatoxins alter the function of host cells by inducing multiple effects through genetic and non-genetic pathways. Epigenetic mechanisms drag major attention towards finding novel and new mechanisms involved in this process. Our present work intends to study the functional expression profile of multiple epigenetic regulators. AFB1 modulates multiple epigenetic regulators like DNA methyltransferases (DMNTs), histones modifying enzymes and polycomb proteins. AFB1 upregulates the expression of DNMTs at gene and protein level in a dose dependent manner. It reduced the histone acetyl transferase (HAT) activity significantly with a remarkable increase in histone deacetylase (HDAC) activity along with an induction in expression of HDACs gene and protein in a dose dependent manner. The gene and protein expression of polycomb repressor proteins B cell specific moloney murine leukemia virus integration site 1 (BMI-1) and enhancer of zeste homolog 2 (EZH2) was significantly over expressed with enhanced trimethylation of H3K27 and ubiquitination of H2AK119. In summary, our results show impact of aflatoxin B1 on multiple epigenetic modulations known to be pivotal in oncogenic processes.AFB1 induces multiple epigenetic modulators in L-132 and HaCaT cell lines.AFB1 affects the expression of DNA methyltransferases and global DNA methylation.Exposure to AFB1 results in a decrease in HAT activity and an upsurge in the HDAC activity.AFB1 modulates the functional expression of polycomb proteins, BMI-1 and EZH2 and increased H3K27me3 and H2AK119Ub.