Background <p>Peritoneal fibrosis(PF) is a major cause of peritoneal membrane failure in peritoneal dialysis(PD) patients, lacking clear occurrence mechanism and reliable non-invasive diagnostic tools. Our study investigated the basic and clinical evidence of fibroblast activation protein (FAP) to detect and quantify PD-associated PF and related potential molecular protein.</p> Methods <p>On the one hand, we evaluated the capacity of FAP in the process of peritoneal fibrosis, including in vitro experiment of human peritoneal mesothelial cells (HPMCs) dataset analysis of GSE162029, and a murine PF model established by daily intraperitoneal injection of 4.25% glucose solution for 2–4 weeks. Finally, ⁶⁸Ga-FAPI-04 PET/CT imaging was performed in animal models and patients. On the other hand, we detected the expression of periostin(POSTN), dipeptidyl peptidase 7(DPP7), and glucagon-like peptide-1 receptor(GLP-1 R) in model animals.</p> Results <p>Increased FAP expression in HPMCs was in a dose-dependent manner (<i>p</i> &lt; 0.01). Transcriptomic analysis confirmed FAP upregulation in glucose-exposed murine peritoneum. In the PF mouse model, with peritoneal transport function deteriorating over time (<i>p</i> &lt; 0.05), histology revealed elevated FAP expression strongly related to indicators of fibrosis. Crucially, ⁶⁸Ga-FAPI-04 PET/CT demonstrated significantly increased peritoneal maximum standardized uptake value(SUV_max) in PF mice compared to controls(<i>p</i> &lt; 0.05). Surprisingly, in patients undergoing dialysis for more than 24 months, a positive ⁶⁸Ga-FAPI-04 PET scan result was inversely correlated with Kt/V(<i>p</i> &lt; 0.05). POSTN and DPP7 were elevated in model animals (<i>p</i> &lt; 0.05).</p> Conclusion <p>FAP expression is significantly elevated during PD-associated PF development and the results of ⁶⁸Ga-FAPI-04 PET/CT, reflected peritoneal functional status and the degree of fibrosis to some extent. Meanwhile, the increase of POSTN and DPP7 were in positive correlation with FAP. The findings represents a promising transformative diagnostic tool for early detection and monitoring of PF and potential mechanism under PF.</p> Trial registration <p>ChiCTR: ChiCTR2300079318. Registered 31 December 2023, <a href="https://www.chictr.org.cn/showproj.html?proj=214233">https://www.chictr.org.cn/showproj.html?proj=214233</a></p>