Objective To investigate the expression level of peptidyl-prolyl isomerase B (PPIB) in esophageal cancer and its relationship with tumor invasion and metastasis, determine the effects of PPIB on the proliferation, migration, and invasion abilities of esophageal cancer cells, and through mechanistic studies analyze its potential feasibility as a novel next-generation sequencing (NGS) technology for application in esophageal cancer. Methods The expression of PPIB in esophageal cancer tissues was analyzed using The Cancer Genome Atlas (TCGA) database. Clinical surgical specimens were collected, and the protein expression level of PPIB was detected by immunohistochemistry to assess its correlation with clinicopathological parameters, particularly lymph node metastasis. Esophageal cancer cell lines with high PPIB expression were selected, and PPIB was knocked down using shRNA. The effect of PPIB silencing on cell proliferation was evaluated by the MTT assay. Changes in cell migration and invasion abilities were examined using Transwell chamber assay, and alterations in PPIB expression and related protein levels were verified by Western blot. A subcutaneous xenograft tumor model was established to evaluate in vivo tumor growth. Hematoxylin and eosin (H&E) staining and immunohistochemical analysis of epithelial-mesenchymal transition-related markers (E-cadherin and Vimentin) were conducted. Data were statistically analyzed using t-tests or analysis of variance (ANOVA). Results Analysis of the TCGA database revealed that the mRNA expression of PPIB in esophageal cancer tissues was significantly higher than that in normal esophageal tissues (P<0.01). Immunohistochemical results further confirmed that the positive expression rate of PPIB in esophageal cancer tissues was significantly higher than that in paired adjacent normal tissues (P<0.05). Clinical correlation analysis indicated that high PPIB expression was closely associated with lymph node metastasis in esophageal cancer patients: the proportion of PPIB high expression was significantly higher in patients with lymph node metastasis than in those without metastasis (P<0.05). In cell experiments, PPIB-knockdown esophageal cancer cell lines were successfully constructed. MTT assays revealed that compared with the control group, the proliferation rate of cells was significantly decreased after PPIB knockdown (P<0.05). Transwell migration and invasion assays showed that silencing PPIB markedly suppressed the migratory and invasive capacities of esophageal cancer cells. In vivo experiments showed that PPIB knockdown slowed tumor growth, reduced final tumor weight, and was accompanied by phenotypic changes including upregulation of E-cadherin and downregulation of Vimentin. Conclusion PPIB is highly expressed in esophageal cancer tissues and is closely associated with tumor invasion and metastasis. Both in vitro functional experiments and in vivo studies confirm that PPIB promotes the proliferation, migration, and invasion of esophageal cancer cells. PPIB may exert a tumor-promoting role in esophageal cancer progression through mechanisms such as inducing epithelial-mesenchymal transition (EMT), preliminarily indicating its potential value as a novel molecular detection biomarker and therapeutic target for esophageal cancer