BackgroundDNA methylation has previously been associated with ischemic stroke, but the specific genes and their functional roles in ischemic stroke remain to be determined. Here we aimed to identify differentially methylated genes that play a functional role in ischemic stroke in a Chinese population.ResultsGenome-wide DNA methylation assessed with the Illumina Methylation EPIC Array in a discovery sample including 80 Chinese adults (40 casesvs. 40 controls) found that patients with ischemic stroke were characterized by increased DNA methylation at six CpG loci (individually located atTRIM6,FLRT2,SOX1,SOX17,AGBL4,andFAM84A, respectively) and decreased DNA methylation at one additional locus (located atTLN2). Targeted bisulfite sequencing confirmed six of these differentially methylated probes in an independent Chinese population (853 casesvs. 918 controls), and one probe (located atTRIM6) was further verified in an external European cohort (207 casesvs. 83 controls). Experimental manipulation of DNA methylation in engineered human umbilical vein endothelial cells indicated that the identified differentially methylated probes located atTRIM6,TLN2,andFLRT2genes may play a role in endothelial cell adhesion and atherosclerosis.ConclusionsAltered DNA methylation of theTRIM6,TLN2,andFLRT2genes may play a functional role in ischemic stroke in Chinese populations.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-023-01520-x.