The regulatory T (Treg) cells play a major role in the control of the autoimmunity and inflammation, and IL-35 has been described as an immunosuppressive cytokine that is mainly produced by CD4(+)FOXP3(+) Treg cells. The aim of this study was to evaluate the serum levels of IL-35 and a single nucleotide polymorphism (SNP), rs3761548, in FOXP3 gene in patients with multiple sclerosis. The blood samples were collected from 140 multiple sclerosis (MS) patients (including 51 untreated and 89 treated patients) and 140 healthy subjects as a control group. The serum levels of IL-35 were measured by ELISA. The DNA was analyzed for SNP rs3761548 in FOXP3 gene using SSP-PCR. There was no significant difference between untreated MS patients and control group regarding the mean serum levels of IL-35, although this parameter was higher in untreated patients. However, the mean serum level of IL-35 in treated MS patients was significantly higher than that in the control group (P < 0.008). The mean serum levels of IL-35 in patients who were treated with interferon-β, methylprednisolone, or with the both interferon-β and methylprednisolone were significantly higher than that in the healthy group (P < 0.01, P < 0.01, and P < 0.2, respectively). The frequencies of AA and AC genotypes at rs3761548 in the FOXP3 gene were significantly higher in MS group as compared with healthy subjects (P < 0.05). The frequency of CC genotype at rs3761548 was significantly lower in the MS group in comparison with healthy control subjects (P < 0.001). Moreover, the frequency of A allele was significantly higher whereas the frequency of C allele was significantly lower in MS patients in comparison to healthy subjects (P < 0.001). The mean serum level of IL-35 was significantly lower in MS patients or healthy subjects with AA genotype as compared with those with CC genotype at rs3761548 in FOXP3 gene (P < 0.01 and P < 0.001, respectively). These results showed higher serum levels of IL-35 in treated MS patients representing that the benefit effects of treatment may in part performed through the upregulation of the IL-35 production. The SNP rs3761548 may influence the susceptibility to MS disease and the serum levels of IL-35.