Background: Esophageal mucosal injury induced by gastroesophageal reflux is a key link to the development of Barrett's esophagus-associated adenocarcinoma. However, the molecular mechanism is still not elucidated. Aims: To investigate the role of differentially expressed genes (DEGs) after stimulating esophageal cells with acid and bile acid in the development of esophageal adenocarcinoma (EAC). Methods: The DEGs were obtained through bioinformatics methods after stimulating esophageal cells with low pH and deoxycholic acid, and GO, KEGG enrichment analysis were performed. Protein-protein interaction (PPI) network was performed to screen the hub genes, and their relationships with prognosis and tumor stage of EAC patients were analyzed. The role of co-expressed genes of GADD45B in EAC was also analyzed. Results: Thirty-one overlapping DEGs were obtained after stimulating esophageal cells with low pH and deoxycholic acid, which mainly enriched in the cytokine-cytokine receptor interaction, transcription factors activity, and regulation of cell proliferation and apoptotic process. High expression of GADD45B was correlated with the survival prognosis and tumor stage of EAC patients. GADD45B and its co-expressed genes were involved in the production of tumor necrosis factor. Conclusions: The high expression of GADD45B induced by acid and bile acid is correlated with the prognosis and tumor stage of EAC patients, and is a potential diagnosis and treatment target for Barrett's esophagus-associated adenocarcinoma.