No coding sequence variants of the gene encoding 5-lipoxygenase-activating protein (ALOX5AP) leading to amino acid substitutions have been identified. Therefore, variants in the ALOX5AP promoter region have received attention recently. The purpose of this study was to explore whether the promoter polymorphism rs17222919 is involved in the etiology of ischemic stroke (IS) in the Chinese Han population. We investigated the rs17222919 polymorphism by TaqMan genotyping in two independent Chinese Han samples: the first comprised 910 IS patients and 925 healthy inhabitants from the northern Henan Province, while the second included 1003 IS patients and 889 healthy controls from the southern Henan Province. Functional characterization of rs17222919 was performed by an in vitro luciferase assay. After adjusting for conventional risk factors, the G allele frequencies in the IS groups were significantly lower than that in the control groups of the two independent Chinese cohorts (19.0% vs. 22.9%, P = 0.004, odds ratio (OR) = 0.792, 95% confidence interval (CI) = 0.675-0.929; 18.8% vs. 22.9%, P = 0.002, OR = 0.782, 95% CI = 0.668-0.915, respectively). This was also observed in the large-artery atherosclerosis (LAA) and stroke of other undetermined etiology (SUE) subtypes (P = 0.019, OR = 0.815, 95% CI = 0.687-0.967; P = 0.021, OR = 0.815, 95% CI = 0.685-0.970, respectively). Additionally, the TG genotype and G allele frequencies were significantly lower in the IS compared with the control group in two female cohorts (P<0.05). Finally, the in vitro luciferase assay demonstrated that the G allele has a significantly lower transcription activity than the T allele (P = 0.031). Our study provides evidence that the promoter single nucleotide polymorphism (SNP) rs17222919 is a potential genetic protective factor for IS in the Chinese Han population.