CXC ligand 16 (CXCL16) is a multifunctional chemokine involved in cell adhesion and chemoattraction as well as in the scavenging of oxidized lipoproteins. Experimental data suggest the roles of CXCL16 in pathogenesis of multiple sclerosis (MS). A181V polymorphism in the human CXCL16 gene has been associated with the clinical course of certain chronic inflammatory diseases. The aim of this study was to analyze the effects of CXCL16 A181V polymorphism on: (1) susceptibility to MS and disease course, (2) peripheral blood mononuclear cells (PBMC) CXCL16 mRNA levels and plasma soluble CXCL16 levels of patients with MS and healthy controls. In this study, 459 MS patients and 303 controls were included. Real-time PCR-based methods were applied for genotyping of CXCL16 A181V and for CXCL16 gene expression analysis. Quantitative sandwich enzyme immunoassay was performed for quantification of plasma soluble CXCL16. CXCL16 AA genotype had a significant protective effect on MS susceptibility in women (OR = 0.53, ±95 % CI = 0.35-0.82, p = 0.004). The V allele-containing genotypes were associated with significantly higher CXCL16 mRNA levels in PBMC of both female (mean factor = 1.81, S.E. = 1.14-2.77, p < 0.01) and male (mean factor = 1.58, S.E. = 1.35-1.73, p < 0.01) controls. No significant association of the CXCL16 polymorphism was established either with soluble CXCL16 plasma levels or with clinical parameters and course of MS. The main finding of this study is gender-specific association of CXCL16 A181V polymorphism with susceptibility to MS in females. The current results should be replicated and validated in the larger sample group.