Background: IL-26 has demonstrated antimicrobial properties, as well as in the degradation of DNA from the Lyme disease spirochete Borrelia burgdorferi (Bb). Additionally, IL-26 can promote macrophage activation and enhance Bb phagocytotic activity. It is unclear if cell-mediated immune responses are modulated through TLR9 signaling when exposed to IL-26 Bb DNA complexes in post-treatment Lyme disease syndrome (PTLDS). Objective: We here aim to explore the effect of IL-26 in human Toll-like receptor (TLR)-9’s activation upon the recognition of Bb DNA. Methods: We utilized a single-receptor cell system, HEK-Dual™ hTLR9 cells, which harbors two reporter plasmids for the NF-κB and IL-8 signaling pathways. Bb DNA was exposed to increasing concentrations of IL-26 in monomeric or dimeric form and then used to stimulate the cells for 4 h. The TLR-9 ligand CpG was used as a control. Results: We observed that NF-κB and IL-8 activation was maximal when the cells were stimulated with Bb DNA that had been treated with 5 µM of IL-26 monomer and 1 µM of IL-26 dimer. The same was observed for IL-8 activation upon CpG stimulation. We observed, however, a decrease in NF-κB activation when treated with either form of IL-26. An NF-κB activation increase did not occur with IL-26-treated TLR9 ligand CpG. Conclusions: Our study shows an enhancement in NF-κB and IL-8 activation upon the recognition of IL-26-treated Bb DNA by TLR9, which suggests an increase in sensing by the TLR9 of Bb DNA when it is in the form of an IL-26-Bb DNA complex. These findings will prompt further studies on the interaction between IL-26 and Bb DNA.