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Background: Esophageal cancer is one of the most common malignant tumors in the digestive system. Comprehensive treatment measures are mainly taken with surgery as the primary supplement to radiotherapy and chemotherapy, but the overall effect is still unsatisfactory. The tripartite motif containing 56 (TRIM56) is a family of TRIM proteins, and it has been found to play an essential role in tumorigenesis and development. We investigated the expression of TRIM56 in esophageal cancer and explored the potential regulatory role of TRIM56 in autophagy in esophageal cancer cells Eca109. Results: In this study, quantitative real-time PCR (qRT-PCR) assay, western blot (WB) and immunofluorescence (IF) were first used to detect the expression of TRIM56 in the Eca109 cells and esophageal cancer tissues. Gene chip analysis showed that the expression of autophagy-related genes LC3 and ATG12 was up-regulated, and regulated autophagy-related signal molecules PIK3CB, AKT2, and FOXO4 expression up-regulation after TRIM56 was down-regulated. Fluorescence immunoassay results showed that after TRIM56 was down-regulated, LC3BII aggregated in the cytoplasm. Conclusion: This study reveals that TRIM56 might inhibit autophagy in the Eca109 esophageal carcinoma cell line.