Certain B cells termed as “regulatory B cells” (Bregs) can suppress the ongoing immune responses and a splenic CD5+CD1dhi Breg subset identified earlier was shown to exert its regulatory functions through secretion of IL-10. Though FasL expression is an alternative mechanism of immune suppression used by B cells, little is known about the FasL expressing CD5+CD1dhi Bregs. In this study, we isolated splenocytes or splenic CD19+ B cells and compared the efficiency of toll-like receptor(TLR)4 ligand (lipopolysaccharide) with TLR9 ligand (CpG), anti-CD40 and TLR9 ligand (CpG) plus anti-CD40 on the FasL expression of splenic CD5+CD1dhi Bregs by flow cytometry. FasL expression in CD5+CD1dhi B cells was rapidly increased after TLR4 ligation. Intriguingly, anti-CD40 and CpG plus anti-CD40 combinations failed to stimulate FasL expression in CD5+CD1dhi B cells although the IL-10 production was up-regulated in this subset. In addition, LPS and other B10-cell inducers increased the expression of surface molecules like CD86 and CD25, which are correlated to the regulatory functions of B cells. Furthermore, NF-κB and NF-AT inhibitors decreased the TLR4-activated FasL expression in CD5+CD1dhi B cells. Then we sorted splenic CD5+CD1dhi Bregs using flow cytometry and found that TLR4-activated CD5+CD1dhi Bregs suppressed the proliferation of CFSE-labeled CD4+ T cells in vitro, which was partly blocked by anti-FasL antibody. In oxazolone-sensitized mice having contact hypersensitivity, FasL expression in splenic CD5+CD1dhi B cells was decreased compared to the control group after TLR4 ligation. Our findings suggest that the regulatory function of CD5+CD1dhi B cells could be partly mediated by Fas-FasL pathway and this FasL expressing CD5+CD1dhi Bregs might participate in the regulation of inflammatory diseases.