Abstract B cells express the innate immune system receptor, Toll like receptor 9 (TLR9), a potent regulator of B cell function, that signals in response to unmethylated CpG sequences in microbial DNA. A dichotomy in the function of the two major classes of CpG-containing oligonucleotides, namely CpG-A and CpG-B, appears to exist with CpG-A restricted to inducing type 1 interferon (type 1 IFN) in innate immune cells and CpG-B to activating B cells to proliferate and produce antibody and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost the innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. We provide evidence that CpG-A activated B cells in vitro in a TLR9 dependent fashion resulting in the production of IL-6, proliferation, and alterations in the expression of a variety of B cell surface markers, albeit in a less robust fashion as compared to CpG-B. Neither CpGB nor CpG-A alone induced B cells to express type 1 IFN. However, when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B induced a robust type 1 IFN response in B cells both in vitro and in vivo. Conversely, CpG-B-DOTAP but not CpGA-DOTAP promoted B cell germinal center responses in vivo and the production of high affinity antibodies. We also provide evidence that difference in the function of CpGs versus DOTAP-associated-CpGs may be related to their intracellular trafficking in B cells since DOTAP enhances the trafficking of CpG-B but not CpG-A to late endosomal compartments. These findings have important implications for the use of CpG-A and CpG-B to augment type 1 IFN production versus B cell responses in vivo.