Two different types of CpG motif‐containing oligonucleotides (CpG ODN) have been described: CpG‐A with high induction of IFN‐α in plasmacytoid dendritic cells; and CpG‐B with little induction of IFN‐α, but potent activation of B cells. In this study, we demonstrate that CpG‐A fail to activate B cells unless plasmacytoid dendritic cells are present. We identified a new set of CpG ODN sequences which induces high levels of IFN‐α in plasmacytoid dendritic cells but remains capable of directly activating B cells. These new CpG ODN (termed CpG‐C) are more potent stimulants of B cells than CpG‐B due to their ability of directly and indirectly (via plasmacytoid dendritic cells) activating B cells. The sequence of CpG‐C combines structural elements of both CpG‐A and CpG‐B. The most potent sequence, M362, contains a 5′‐end ‘TCGTCG‐motif’ and a ‘GTCGTT‐motif’, both of which are present in CpG‐B (ODN 2006); a palindromic sequence characteristic for CpG‐A (ODN 2216); but no poly G motif required for CpG‐A. In conclusion, we defined the first CpG‐containing sequences that potently activate both TLR9‐expressing immune cell subsets in humans, the plasmacytoid dendritic cell and the B cell. CpG‐C may allow for improved therapeutic immuno‐modulation in vivo.