The regulation of gene expression plays a pivotal role in the complex metamorphic process of flatfishes, which oversees the dynamic alterations that occur during their transition from a pelagic to a benthic way of life. Flatfish metamorphosis is characterized by substantial modifications in both form and function, which are initiated by thyroid hormones. Epigenetic mechanisms play a vital role in the regulation of gene expression during this transformative process. This study examines the molecular mechanisms underlying flatfish metamorphosis by integrating multi-omics data that provide information on chromatin status, DNA methylation profile, and gene expression at three key stages (pre-metamorphosis, climax and post-metamorphosis) in the turbot (Scophthalmus maximus) brain, a complex organ that plays a critical role in the regulation of this intricate process. The analysis of DNA methylation patterns revealed major epigenetic dynamic changes during the different metamorphosis stages. Specifically, the methylation levels exhibited a typical bimodal distribution in the pre-metamorphic stage, transitioned to intermediate levels of methylation (around 50%) during the climax stage, and reverted to a bimodal distribution in the post-metamorphic stage. Notably, DMRs were identified in regions of open chromatin that colocalized with CpG islands. Moreover, our results indicate an inverse relationship between DNA methylation and transcriptional activity in regions near the transcription start sites (TSSs), where high levels of methylation correspond to low expression and low levels of methylation corresponds to high gene transcription. This study thereby elucidates the regulatory impact of methylation on gene expression during the metamorphosis process in the flatfish brain.