Abstract: Objective To investigate the malignant progression and molecular mechanism of KHSRP regu- lating esophageal squamous cell carcinoma (ESCC) through the JAK1/STAT3 signaling axis. Methods Tumor tissues and adjacent non-tumor tissues were collected from 72 patients with ESCC. Human normal esophageal epithelial cells (Het-1A) and multiple ESCC cell lines (EC-9706, TE-7, KYS-450, FLO- 1, SK-GT- 4, BE-3) were cultured. The expression level of KHSRP in the cells was detected using real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Through lentiviral transfection technology, stable KHSRP-knockdown EC- 9706 and SK-GT-4 cell models (sh-KHSRP group), as well as stable KHSRP-overexpressing BE-3 and KYS-450 cell models (KHSRP group), were established, and corresponding negative control groups (sh-NC group and Vec- tor group) were also established. Cell proliferation, migration, and invasion abilities were assessed using the cell counting kit-8 (CCK-8) assay, Transwell migration assay, and Transwell invasion assay, respectively. A total of 62 male BALB/C nude mice aged 4 to 6 weeks were selected for the experiments. Thirty-two nude mice with subcuta- neous tumor-loading experiments were randomly divided into four groups:sh-KHSRP 1 group, sh-NC 1 group, KHSRP 1 group, and Vector 1 group, with 8 mice in each group. Thirty nude mice with tail vein injection for lung metastasis model experiments were randomly divided into four groups:sh-KHSRP 2 group (n=7), sh-NC 2 group (n=7), KHSRP 2 group (n=8), and Vector 2 group (n=8). Tumor volume and mouse body weight were meas- ured regularly. After sampling at the experimental endpoint, immunohistochemical analysis was performed to further validate the biological functions and potential molecular mechanisms of KHSRP in ESCC. The expression levels of JAK/STAT and KHSRP proteins in stably transfected cells were detected using western blotting. Results The re- sults of RT-qPCR revealed that, compared with human normal esophageal epithelial cells, the expression of KHSRP was significantly elevated in ESCC cell lines (EC-9706, TE-7, KYS-450, FLO-1, SK-GT-4, BE-3) (P<0.001). The CCK-8 assay demonstrated that, compared with the sh-NC group, the proliferative activity of EC-9706 and SK-GT-4 cells in the sh-KHSRP group was significantly reduced (P<0.05); compared with the Vector group, the proliferative activity of BE-3 and KYS-450 cells in the KHSRP group was significantly increased (P<0.001). The Transwell assay further indicated that, compared with the sh-NC group, the migration and invasion abilities of EC-9706 and SK-GT-4 cells in the sh-KHSRP group were significantly inhibited, with a marked decrease in the number of cells passing through the membrane (P<0.05); compared with the Vector group, the migration and in- vasion abilities of BE-3 and KYS- 450 cells in the KHSRP group were significantly enhanced, with a notable in- crease in the number of cells passing through the membrane (P<0.05). The results of the subcutaneous tumor- loading experiment showed that, compared with the sh-NC 1 group, the tumor volume and weight in the sh-KHSRP 1 group were significantly reduced (P=0.041, P=0.006); compared with the Vector 1 group, the tumor volume and mass in the KHSRP 1 group were significantly increased (P=0.038, P=0.003). The tail vein injection lung metastasis model experiment revealed that, compared with the sh-NC 2 group, the number of metastatic nodules in the sh-KHSRP 2 group was decreased (P=0.019, P=0.014); compared with the Vector 2 group, the num- ber of metastatic nodules in the KHSRP 2 group was significantly increased (P=0.008, P=0.005). The results of the signaling pathway experiment showed that, compared with the sh-NC group, the expression levels of JAK1 and STAT3 in the sh-KHSRP group were significantly downregulated (P<0.001); compared with the Vector group, the expression levels of JAK1 and STAT3 in the KHSRP group were significantly upregulated (P<0.001). Conclusions KHSRP is upregulated in ESCC and can positively regulate the JAK1/STAT3 signa- ling axis, potentially promoting the malignant progression of metastasis in ESCC.