Stimulation of endosomal associated TLR9 by DNA plays an important role in activation of plasmacytoid dendritic cells (pDCs) and autoreactive B cells and may contribute to the pathogenesis of systemic lupus erythematosus (SLE). HMGB1 is a DNA binding protein that is liberated either from cells undergoing necrosis or following cytokine stimulation. Here we show that HMGB1 forms a high affinity complex with CpG-A oligodeoxynucleotides and augments the production of IFN-a from murine pDCs. We also show that HMGB1 is indeed a component of naturally formed DNA containing chromatin complexes. IFNa induced by either the exogenously formed HMGB1/CpG-A complex or naturally formed complex is TLR9 dependent and is inhibited by HMGB1 and RAGE antagonists. The complex without HMGB1 induces significantly less IFNa. HMGB1-RAGE interactions are required for activation of autoreactive B cells following stimulation with DNA immune complexes and are critical in the regulation of type I interferon gene induction by DNA complexes present in lupus plasma. Our data provide a novel mechanism by which HMGB1 confers potent stimulatory activity to DNA through a RAGE dependent mechanism and may contribute to the pathogenesis of lupus.