The levels of serum S100B were elevated in patients with ischemic stroke (IS), which may be a novel biomarker for diagnosing IS. The aim of this study was to investigate the association of S100B polymorphisms and serum S100B with IS risk. We genotyped the S100B polymorphisms rs9722, rs9984765, rs2839356, rs1051169 and rs2186358 in 396 IS patients and 398 controls using polymerase chain reaction-single base extension (SBE-PCR). Serum S100B levels were measured by enzyme-linked immunosorbent assay (ELISA). Rs9722 was associated with an increased risk of IS (AA vs. GG: adjusted OR = 2.172, 95% CI, 1.175-4.014, P = 0.013; dominant: adjusted OR = 1.507, 95% CI, 1.071-2.123, P = 0.019; recessive: adjusted OR = 1.846, 95% CI, 1.025-3.323, P = 0.041; additive: adjusted OR=1.371, 95% CI, 1.109-1.694, P = 0.003). The A-C-C-C-A haplotype was associated with an increased risk of IS (OR = 1.325, 95% CI, 1.035-1.696, P = 0.025). In addition, individuals carrying the rs9722 GA/AA genotypes had a higher serum S100B compared with the rs9722 GG genotype in IS patients (P = 0.018). Our results suggest that the S100B gene rs9722 polymorphism may contribute to the susceptibility of IS, probably by promoting the expression of serum S100B.