<p>Cancer-associated fibroblasts (CAFs) secrete and synthesize fibroblast activation protein (FAP), which could promote proliferation and immunosuppression of multiple cancers including esophageal squamous cell carcinoma (ESCC). CXCL12/CXCR4 signaling could be revitalized by CAFs in cancer cells. Nevertheless, the significance of this interaction in ESCC has yet to be elucidated. Herein, we investigated whether FAP⁺ CAF cells could promote ESCC cells proliferation, migration and regulate immunity through the CXCL12/CXCR4 pathway in vitro and in vivo. The protein expression level of FSP1, FAP, CD8+ and Ki-67 in different sample was estimated by IHC and western blot. qPCR was used to quantify the mRNA level of FSP1, FAP, CD8+ and Ki-67 in different sample. The cell viability, proliferation, migration and invasion of different sample were evaluated by CCK-8, EdU staining, wound healing assay and Transwell assay, respectively. The ELISA was carried out to measure the protein level of IFN-γ, TNF-α, GZMB and IL-2. ESCC xenograft mice model was established to assess the impact of FAP+ CAF. FSP1, FAP, CD8+ and Ki-67 are greatly up-regulated in hESCC tissues. Through CXCL12/CXCR4 axis, FAP-positive CAF was capable of promoting the cell proliferation, migration and invasion of ESCC tumor cells and preventing the CD8+ T cells from secreting cytokine. Blocking this signaling with selective CXCR4 antagonist could counteract the effects caused by high-expression of FAP. FAP+ CAFs could inhibit the occurrence and development of tumors. These results indicated that FAP-positive CAF have an impact on cell proliferation migration and immunoregulation of ESCC through the CXCL12/CXCR4 axis.</p>