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Innate immune signals that promote B cell responses in gene transfer are generally ill-defined. In this study, we evaluate the effect of activating endosomal Toll-like receptors 7, 8, and 9 (TLR7, TLR7/8, and TLR9) on antibody formation during muscle-directed gene therapy with adeno-associated virus (AAV) vectors. We examined whether activation of endosomal TLRs, by adenine analog CL264 (TLR7 agonist), imidazolquinolone compound R848 (TLR7/8 agonist), or class B CpG oligodeoxynucleotides ODN1826 (TLR9 agonist), could augment antibody formation upon intramuscular administration of AAV1 expressing human clotting factor IX (AAV1-hFIX) in mice. The TLR9 agonist robustly enhanced antibody formation by the 1st week, thus initially eliminating systemic hFIX expression. By contrast, the TLR7 and TLR7/8 agonists did not markedly promote antibody formation, or significantly reduce circulating hFIX. We concurrently investigated the effects of these TLR agonists during muscle gene transfer on mature B cells and dendritic cells (DCs) in the draining lymph nodes including conventional DCs (CD11b + or CD8α + cDCs), monocyte-derived dendritic cells (moDCs), and plasmacytoid dendritic cells (pDCs). Only TLR9 stimulation caused a striking increase in the frequency of moDCs within 24 h. The TLR7/8 and TLR9 agonists activated pDCs, both subsets of cDCs, and mature B cells, whereas the TLR7 agonist had only mild effects on these cells. Thus, these TLR ligands have distinct effects on DCs and mature B cells, yet only the TLR9 agonist enhanced the humoral immune response against AAV-expressed hFIX. These new findings indicate a unique ability of certain TLR9 agonists to stimulate B cell responses in muscle gene transfer through enrichment of moDCs.