Inflammation is a pathophysiological response of innate immunity to infection or tissue damage. This response is among others triggered by factors released by damaged or dying cells, termed damage-associated molecular pattern (DAMP) molecules that act as danger signals. DAMPs interact with pattern recognition receptors (PRRs) to contribute to the induction of inflammation. However, how released peroxiredoxins (PRDXs) are able to activate PRRs, such as Toll-like receptors (TLRs), remains elusive. Here, we used force-distance curve-based atomic force microscopy to investigate the molecular mechanisms by which extracellular human PRDX5 can activate a proinflammatory response. Single-molecule experiments demonstrated that PRDX5 binds to purified TLR4 receptors, on macrophage-differentiated THP-1 cells, and on human TLR4-transfected CHO cells. These findings suggest that extracellular PRDX5 can specifically trigger a proinflammatory response. Moreover, our work also revealed that PRDX5 binding induces a cellular mechanoresponse. Collectively, this study provides insights into the role of extracellular PRDX5 in innate immunity.