The cytotoxic T lymphocyte antigen 4 gene (CTLA4) is a critical regulator of T-cell activation and it is an important therapeutic target for cancer and autoimmune diseases. Here, we analyzed the genomic regulation of CTLA4 gene expression in order to identify single nucleotide polymorphisms (SNPs) that affect its expression and splicing, and to assess their association with Multiple Sclerosis (MS). We analyzed 152 healthy subjects and 146 patients with MS, of which 52 controls and 51 patients were used for gene expression analysis. We genotyped 17 SNPs in the CTLA4 gene using the SNaPshot Multiplex Kit, and in addition gene expression of the soluble (sCTLA4) and full length (flCTLA4) isoforms was quantified by real-time PCR, while protein levels of sCTLA4 were measured by ELISA. We found that the SNPs at -1577, +6230, +10242, +10717 and +12310 influence CTLA4 expression and the combination of the -1577 GG and +6230 GG genotypes provokes the strongest decrease in CTLA4 gene expression. We found that the SNP at -658 only acted as a regulatory SNP in patients with MS, suggesting the existence of epigenetic changes due to this disease. We also identified a decrease in CTLA4 gene expression levels in patients receiving chemotherapy, although no association was observed between MS and any of the polymorphisms studied. In conclusion, we have identified several SNPs in the CTLA4 gene and studied their influence on its genetic regulation. The involvement of CTLA4 in the pathogenesis of MS may be subtle and related to the functional changes in its pathway rather than predisposing genetic polymorphisms.