Introduction <p>Post-traumatic urethral stricture, characterized by hypertrophic scar formation, poses significant therapeutic challenges. Infiltration of neutrophil extracellular traps (NETs) is a prominent feature of post-traumatic urethral microenvironment, but their roles remain unknown.</p> Objectives <p>To investigate the mechanisms by which NETs mediate urethral fibrosis and explore potential therapeutic strategies targeting NETs.</p> Methods <p>Single-cell RNA sequencing was performed on urethral tissues from New Zealand rabbits with urethral injury to reveal the cell atlas of post-traumatic inflammatory microenvironment. The level of was assessed in both clinical and animal samples using immunohistochemistry and ELISA. In vitro, NETs were co-cultured with urethral fibroblasts (UFBs) to assess their effects and the mechanism underlying. Additionally, animal models of urethral injury were treated with NETs inhibitors to assess the effects of NETs on urethral scar formation.</p> Results <p>We constructed a single-cell atlas of the post-traumatic urethral microenvironment and found that NETs infiltration was correlated closely with the development of urethral fibrosis. NETs activated UFBs via the TLR9/NF-κB/Smad3 pathway, promoting collagen synthesis and IL-8 secretion, which further recruited neutrophils, creating a positive feedback loop. In vivo, DNase I and AT791 significantly reduced NET levels, suppressed fibroblast activation, and mitigated collagen deposition, effectively reducing stricture formation.</p> Conclusion <p>NETs drive post-traumatic urethral fibrosis by activating fibroblasts and amplifying inflammation. Targeting NETs or their uptake via TLR9 inhibition may offer a promising therapeutic strategy for urethral stricture.</p> Graphical abstract <p>NETs drive post-traumatic urethral fibrosis by activating fibroblasts and amplifying inflammation. Targeting NETs or their uptake via TLR9 inhibition may offer a promising therapeutic strategy for urethral stricture.</p>