Multiple sclerosis (MS) is a devastating inflammatory disease of the brain and spinal cord that is thought to result from an autoimmune attack directed against antigens in the central nervous system. The aim of this first-in-man trial was to assess the feasibility, safety, and tolerability of a tolerization regimen in MS patients that uses a single infusion of autologous peripheral blood mononuclear cells chemically coupled with seven myelin peptides (MOG1–20, MOG35–55, MBP13–32, MBP83–99, MBP111–129, MBP146–170, and PLP139–154). An open-label, single-center, dose-escalation study was performed in seven relapsing-remitting and two secondary progressive MS patients who were off-treatment for standard therapies. All patients had to show T cell reactivity against at least one of the myelin peptides used in the trial. Neurological, magnetic resonance imaging, laboratory, and immunological examinations were performed to assess the safety, tolerability, and in vivo mechanisms of action of this regimen. Administration of antigen-coupled cells was feasible, had a favorable safety profile, and was well tolerated in MS patients. Patients receiving the higher doses (>1 × 109) of peptide-coupled cells had a decrease in antigen-specific T cell responses after peptide-coupled cell therapy. In summary, this first-in-man clinical trial of autologous peptide-coupled cells in MS patients establishes the feasibility and indicates good tolerability and safety of this therapeutic approach.