IFN-β-1b is a first-line immunomodulatory therapy for relapsing-remitting multiple sclerosis (RR MS). However, its effects on B cells have not been characterized. In vitro studies of B cells derived from RR MS patients revealed that IFN-β-1b decreases B cells' stimulatory capacity, as detected by inhibition of the Ag-specific T cell proliferative response upon Ag presentation by IFN-β-1b-treated B cells. Our study has identified that IFN-β-1b inhibited B cells' stimulatory capacity in RR MS patients and healthy controls through the suppression of CD40 and CD80 expression, whereas the MHC class I and II expression was not changed. IFN-β-1b in vitro treatment inhibited B cell secretion of IL-1β and IL-23 and induced IL-12 and IL-27. Supernatants transferred from IFN-β-1b-treated B cells inhibited Th17 cell differentiation, as they suppressed gene expression of the retinoic acid-related orphan nuclear hormone receptor C and IL-17A and secretion of IL-17A. In addition, IFN-β-1b induced B cells' IL-10 secretion, which may mediate their regulatory effect. Studies of B cells derived from RR MS patients treated with recombinant s.c. injected IFN-β-1b revealed that they induced a significantly lower proliferative response in allogenic MLR than the B cells from untreated patients. Further confirming the IFN-β-1b in vitro-induced changes in B cell cytokine secretion, B cells derived from the IFN-β-1b-treated patients secreted significantly lower levels of IL-1β and IL-23 and higher levels of IL-12 and IL-27 in comparison with the B cells derived from untreated patients. We conclude that IFN-β-1b exerts its therapeutic effects in part by targeting B cells' functions that contribute to the autoimmune pathogenesis of RR MS.