Purpose <p>Esophageal squamous cell carcinoma (ESCC) is aggressive with a poor prognosis. The tumor microenvironment (TME) significantly affects tumor progression and therapy resistance. Previous work has shown that fibroblasts in metastatic lymph nodes can confer cisplatin resistance to ESCC cells via PI16 (peptidase inhibitor 16). This study investigates the role of fibroblast-derived PI16 in the ESCC TME.</p> Methods <p>Public single-cell RNA sequencing (scRNA-seq) data for ESCC were analyzed. A cell co-culture assay was performed to evaluate regulatory T cells (Tregs) differentiation from naïve CD4⁺ T cells. Immunoprecipitation and mass spectrometry examined PI16’s mechanism in Treg differentiation. In vitro and in vivo assays were conducted to explore fibroblast-derived PI16’s function. Additionally, multiplex fluorescent immunohistochemistry (mfIHC) was performed.</p> Results <p>Analyses of the scRNA-seq dataset (GSE203115) reveal that fibroblasts can be classified into PI16 ⁺ and PI16^- subclusters based on PI16 expression levels. PI16 induces Treg differentiation from naïve CD4⁺ T cells through a DOCK2-dependent mechanism. Treatment with a DOCK2 inhibitor significantly inhibits PI16-induced Treg differentiation and increases Teff cell infiltration in vivo. Moreover, upregulation of PI16 in the tumor stroma is associated with poorer long-term survival outcomes in patients with ESCC.</p> Conclusions <p>PI16⁺ fibroblasts promote the differentiation of Tregs from naïve CD4⁺ T cells through interaction with DOCK2. Upregulation of PI16 in the tumor stroma is associated with poorer long-term survival outcomes in patients with ESCC. Given the accumulating evidence on the therapeutic impact of targeting the TME, PI16⁺ fibroblasts emerge as a promising novel therapeutic target to overcome tumor immune suppression.</p>