Highlight: • Pretreatment with 5-azaC activates the antioxidant system, reducing reactive oxygen species (ROS) and alleviating salt stress in kenaf.• 5-azaC pretreatment can increase the content of chlorophyll, starch, soluble sugars, and adenosine triphosphate (ATP) in kenaf seedlings under salt stress.• MethylRAD methylome analysis showed 5-azaC pretreatment significantly decreased genome-wide DNA methylation level under salt stress.• 5-azaC pretreatment regulated differentially methylated genes (DMGs) and metabolic pathways played essential roles in kenaf salt tolerance.• VIGS silencing of DNA demethylase gene, of repressor of silencing 1 (HcROS1), reduced kenaf salt tolerance capacity.