A hallmark of systemic lupus erythematosus (SLE) is the consistent production of various auto-antibodies by auto-reactive B cells. Interferon-α (IFN-α) signaling is highly activated in SLE B cells and plays a vital role in the antibody response by B cells. Previous studies have shown that CD180-negative B cells, which are dramatically increased in SLE patients, are responsible for the production of auto-antibodies. However, the association between CD180 and IFN-α signaling remains unknown. In the present study, we explored the effect of CD180 on regulating the activation of IFN-α signaling in B cells. We found that the number of CD180-negative B cells was increased in MRL/Mp-Fas(lpr/lpr) lupus-prone mice compared with wild-type mice. Phenotypic analysis showed that CD180-negative B cells comprised CD138+ plasmablast/plasma cells and GL-7+ germinal center (GC) B cells. Notably, ligation of CD180 significantly inhibited the IFN-α-induced phosphorylation of signal transducer and activator of transcription 2 (STAT-2) and expression of IFN-stimulated genes (ISGs) in a Lyn-PI3K-BTK-dependent manner in vitro. Moreover, ligation of CD180 could also inhibit IFN-α-induced ISG expression in B cells in vivo. Furthermore, the Toll-like receptor 7 and Toll-like receptor 9 signaling pathways could significantly downregulate CD180 expression and modulate the inhibitory effect of CD180 signaling on the activation of IFN-α signaling. Collectively, our results highlight the close association between the increased proportion of CD180-negative B cells and the activation of IFN-α signaling in SLE. Our data provide molecular insight into the mechanism of IFN-α signaling activation in SLE B cells and a potential therapeutic approach for SLE treatment.