The surface Toll-like receptor 9 (sTLR9) has been identified on the surface of the B cells and was presumed to be a negative regulator of B cell responses. CpG ODN, a TLR9 agonist, has been successfully used as an adjuvant of hepatitis B vaccine to enhance antibody responses. However, it is unknown whether the sTLR9 is involved in regulating the activation and maturation of B cells in the antibody responses induced by CpG ODN-adjuvanted vaccines. In this study, we immunized mice with hepatitis B vaccine adjuvanted by CpG ODN (CpG 5805) and found that CpG 5805 enhanced the antibody response to vaccine and meanwhile down-regulated the sTLR9 levels on B cells. With antibody feeding assay and flow cytometry analysis, we further found that CpG 5805 induced a movement of the sTLR9 in B cells, internalized first and then mobilized to endosomes. Accompanied with the movement, CD80, CD86, CD40, and MHC II molecules were significantly up-regulated on the B cells. Interestingly, the B cells with internalized sTLR9 enlarged morphologically, and the sTLR9 levels were obviously lower and CD40 levels were obviously higher on the enlarged B cells. Together, the data presented here uncover that CpG ODN can induce the mobilization and relocation of sTLR9 in B cells, thereby triggering the B cell vigor by relieving the negative regulatory effect of sTLR9 on B cells, which may be one of the mechanisms for CpG ODN acting as a vaccine adjuvant to enhance the antibody response.