Our study aimed to investigate the associations between CXCL12 rs1029153, rs1801157, and rs2297630 single-nucleotide polymorphisms (SNPs), CXCL12 protein levels, MS prevalence, and clinical parameters. This study included 250 individuals diagnosed with MS and 250 sex- and age-matched healthy control individuals from Lithuania. The SNPs were genotyped with real-time PCR-based assays. The CXCL12 protein concentration was evaluated in serum using the ELISA method. Of the studied CXCL12 SNPs, we found that the rs1801157 CT genotype in the males was associated with 2.3 times reduced MS odds when compared with the CC genotype according to the overdominant and codominant models (p = 0.011 and p = 0.012, respectively). There was a tendency, which did not reach adjusted statistical significance, for a lower CXCL12 protein concentration in the healthy individuals with the rs1801157 CT genotype (p = 0.028). Sensory symptoms were rarer in the women with the rs1801157 TT genotype (p = 0.004); however, this genotype was also associated with a shorter MS disease duration (p = 0.007). CXCL12 rs1801157 was associated with reduced odds of MS occurrence in the male individuals. In women, rs1801157 was associated with a sensory symptom prevalence.