Background:Glucocorticoid receptor (GR) mediated corticosterone-induced fatty liver syndrome (FLS) in the chicken by transactivation of Fat mass and obesity associated gene (FTO), leading to demethylation of N6- methyladenosine (m6A) and post-transcriptional activation of lipogenic genes. Nutrition is considered the main cause of FLS in the modern poultry industry. Therefore, this study was aimed to investigate whether GR and m6A modification are involved in high-energy and low protein (HELP) diet-induced FLS in laying hens, and if true, what specific m6A sites of lipogenic genes are modified and how GR mediates m6A-dependent lipogenic gene activation in HELP diet-induced FLS in the chicken. Results:Laying hens fed HELP diet exhibit excess (P<0.05) lipid accumulation and lipogenic genes activation in the liver, which is associated with significantly increased (P<0.05) GR expression that coincided with global m6A demethylation. Concurrently, the m6A demethylase FTO is upregulated (P<0.05), whereas the m6A reader YTHDF2 is downregulated (P<0.05) in the liver of FLS chickens. Further analysis identifies site-specific demethylation (P<0.05) of m6A in the mRNA of lipogenic genes, including FASN, SREBP1 and SCD. Moreover, GR binding to the promoter of FTO gene is highly enriched (P<0.05), while GR binding to the promoter of YTHDF2 gene is diminished (P<0.05). Conclusions:These results implicate a possible role of GR-mediated transcriptional regulation of m6A metabolic genes on m6A-depenent post-transcriptional activation of lipogenic genes and shed new light in the molecular mechanism of FLS etiology in the chicken.