Helicobacter pylori (H. pylori) are one of the most common, well-known pathogenic agents in the development of peptic ulcers. Some investigators have shown a relationship between H. pylori and Multiple Sclerosis (MS). However, this relationship is controversial. The aim of this study was to determine the association between H. pylori infection and MS. In a prospective case-control study, we studied 78 patients with MS and 123 Healthy Blood Donors (HBDs) for bacterial DNA detection and antibody assay. DNA extracted from samples (serum and saliva) and Real-time PCR was employed to detection of H. pylori genome. The present of anti H. pylori CagA and VacA Immunoglobulin G (IgG) were measured in serum by Western blot technique. We found H. pylori DNA in both samples of the 32.05% (25/78) and 32.52% (40/123) of patients and HBDs respectively (p = NS). Furthermore, anti H. pylori IgG for both antigens were detected in 21.95% (27/123) of HBDs in contrast with 25.64% (20/78) of patients (p = NS). Moreover, genome copy number of H. pylori was not significantly change in patients (140 copies/mL) and HBDs (147 copies/mL). We didn’t see significant correlation between H. pylori infection in both groups, But H. pylori CagA/VacA-IgG was found in patient quite more than HBDs (p<0.05) and this patients showed more positively for serum H. pylori genome. Although, these results indicate a lack of connection between the Helicobacter pylori infection and multiple sclerosis, the role of immune response against H. pylori in the modulation of MS requires further study.