Introduction: Low vitamin D levels have been recognised as an important risk factor for autoimmune diseases, including multiple sclerosis (MS). MS is a multifactorial disease, the pathogenesis of which contributes both to genetic and environmental factors. Polymorphisms in genes codifying molecules involved in vitamin D homeostasis have been associated with hypovitaminosis D. However, the influence of polymorphisms of Klotho, which codify a protein with a pivotal role in vitamin D metabolism, have never been investigated. The aim of this study was to evaluate the association among genetic variants of Klotho, namely rs1207568 and rs9536314, serum 25(OH)D 3 levels, and multiple sclerosis (both risk and disease progression). Material and methods: 107 patients with MS and 133 healthy controls were enrolled in this study. Serum 25(OH)D 3 levels and genotyping of Klotho SNPs were evaluated in all participants by high-performance liquid chromatography and real-time polymerase chain reaction, respectively. Results: Allelic and genotypic frequencies did not differ between patients and controls. Concerning rs1207568, we found a trend toward lower serum 25(OH)D 3 levels in MS patients with A allele (mutant), both in heterozygosis (AG) and in homozygosis (AA), in comparison to MS patients with G allele in homozygosis (GG) (AG + AA 20.5 ±6.3 μg/l; GG 22.5 ±7.5 μg/l, p = 0.07). Conclusions: Our findings did not identify a role of Klotho in the genetic susceptibility to MS.