Background: B cells are key pathogenic effectors in multiple sclerosis (MS) and several therapies have been designed to restrain B cell abnormalities by directly targeting this lymphocyte population. Objectives: Moving from our data showing a Toll-like receptor (TLR)7-driven dysregulation of B cell response in relapsing–remitting multiple sclerosis (RRMS) and having found a low serum level of Thymosin-α1 (Tα1) in patients, we investigated whether the addition of this molecule to peripheral blood mononuclear cells (PBMCs) would influence the expansion of regulatory B cell subsets, known to dampen autoimmune inflammation. Methods: Serum Tα1 level was measured by enzyme immunoassay. Cytokine expression was evaluated by Cytometric Bead Array (CBA), enzyme-linked immunosorbent assay (ELISA), and real-time reverse transcription polymerase chain reaction (RT-PCR). B cell subsets were analyzed by flow cytometry. Results: Tα1 pre-treatment induces an anti-inflammatory status in TLR7-stimulated RRMS PBMC cultures, reducing the secretion of pro-inflammatory interleukin (IL)-6, IL-8, and IL-1β while significantly increasing the regulatory IL-10 and IL-35. Indeed, Tα1 treatment enhanced expansion of CD19⁺CD24⁺CD38^hi transitional-immature and CD24^low/negCD38^hi plasmablast-like regulatory B cell subsets, which likely inhibit both interferon (IFN)-γ and IL-17 production. Conclusion:: Our study reveals a deficient ability of B cells from MS patients to differentiate into regulatory subsets and unveils a novel anti-inflammatory and repurposing potential for Tα1 in MS targeting B cell response.