Previous studies have shown that CpG-SNPs might have influence on gene function via allele-specific DNA methylation (ASM). However, association study between DNA methylation and the promoter CpG-SNPs in ALOX5AP gene with IS has not been reported. The present study aims to explore the relationship among CpG-SNPs, methylation levels, and messenger RNA (mRNA) expression levels of ALOX5AP gene. Firstly, we made a two-stage association study to identify a potential associated CpG-SNP (rs4073259) by SNaPshot genotyping approach (P = 0.015, OR = 0.672, 95% CI 0.487–0.927; P = 0.035, OR = 0.809, 95% CI 0.664–0.985, respectively). In addition, the methylation levels of 17 CpG sites located in the promoter of ALOX5AP were tested by MethylTarget sequencing. The methylation level of GG genotype carriers is significantly higher than those with the AG and AA genotypes (P