We tested the hypothesis that altered Toll-like receptor (TLR) signaling may be involved in early stages of type 1 diabetes (T1D). To do so, we analyzed TLR-induced interleukin (IL)-1β and IL-6 responses in freshly isolated peripheral blood mononuclear cells (PBMNCs) from seropositive compared with seronegative subjects. Similar frequencies of myeloid dendritic cells (mDCs), plasmacytoid DCs (pDCs), and monocytes were observed in seropositive and seronegative subjects. Subjects with autoantibodies had increased proportions of monocytes expressing IL-1β ex vivo. Activating PBMNCs with TLR3, TLR4, or TLR7/8 agonists in vitro led to increased percentages of IL-1β-expressing monocytes and mDCs from seropositive versus seronegative subjects. TLR ligation also resulted in a diminished IL-6 response in seropositive individuals as lower frequencies of IL-6-expressing monocytes and mDCs were induced. The dysregulated TLR-induced IL-1β and IL-6 pathways were more readily detectable in children aged <11 years and from 11 to <21 years, respectively, and did not involve altered HbA(1c) or the presence of one or more autoantibodies. Finally, subjects with autoantibodies had lower amounts of serum chemokine (C-X-C motif) ligand 10 compared with autoantibody-negative subjects. Our data may imply that alterations in innate immune pathways are detectable in genetically susceptible individuals and could be linked with the early course of T1D.