AbstractDNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium and displayed a slightly flavescent conidial pigmentation compared with the normal yellow of the wild-type strain when grown on agar. The ΔdmtA lines exhibited decreased conidiation and aflatoxin (AF) biosynthesis, compared with the wild-type line, suggesting that the DmtA knock-out affected the transcriptional level of genes in the AF cluster. In particular, sclerotia development and host colonization were altered in the dmtA null mutants. Green fluorescent protein tagging at the C-terminus of DmtA showed that DmtA localized to the nucleus and cytoplasm. DNA methylation content measurements in the dmtA mutants revealed no widespread DNA methylation in the mutants or wild-type lines. Thus, our findings suggest that DmtA, apart from being a C-5 cytosine methyltransferase in A. flavus, contributes to asexual development, aflatoxin biosynthesis, sclerotial production and virulence.