The success of using immune checkpoint inhibitors to treat cancers implies that inhibiting an immunosuppressive cytokine, such as TGF-β2, could be a strategy to develop novel adjuvants for microbial vaccines. To develop nucleic acid based TGF-β2 inhibitors, we designed three antisense oligonucleotides, designated as TIO1, TIO2, and TIO3, targeting the conserve regions identical in human and mouse TGF-β2 mRNA 3′-untranslated region. In cultured immune cells, TIO3 and TIO1 significantly reduced the TGF-β2 mRNA expression and protein production. In mice, the TIO3 and TIO1, when formulated in various microbial vaccines, significantly enhanced the antibody response to the vaccines, and the TIO3-adjuvanted influenza virus vaccine induced effective protection against the influenza virus challenge. In the immunized mice, TIO3 formulated in microbial vaccines dramatically reduced surface-bound TGF-β2 expression on CD4 T cells and CD19 B cells in the lymph node (LN) cells and spleen cells; up-regulated the expression of CD40, CD80, CD86, and MHC II molecules on CD19 B cells and CD11c dendritic cells; and promoted IFN-γ production in CD4 T cells and CD8 T cells in the LN cells. Overall, TIO3 or TIO1 could be used as a novel type of adjuvant for facilitating the microbial vaccines to elicit more vigorous and persistent antibody response by interfering with TGF-β2 expression.©2020 Society for Leukocyte Biology.