ABSTRACT: Astronauts suffer from inflammatory changes induced by microgravity during space flight. Microgravity can significantly affect the inflammatory response of various cell types and multiple systems of the human body, such as cardiovascular system, skeletal muscle system, and digestive system. The aim of this research was to identify the key genes and pathways of gastric mucosa affected by microgravity. Human gastric mucosal epithelial GES-1 cells were cultured in a rotary cell culture system (RCCS) bioreactor to simulate microgravity. The gene expression profiles of GES-1 cells were obtained using Illumina sequencing platform and differentially expressed genes were identified by DESeq2 software, then Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Subsequently, a protein-protein interaction (PPI) network was constructed. Compared with a normal gravity (NG) group, a total of 943 DEGs, including 192 downregulated genes and 751 upregulated genes, were identified. These DEGs were associated with findings that included response to interleukin-1, positive regulation of inflammatory response, and positive regulation of neuroinflammatory response. Furthermore, these DEGs were mainly enriched in herpes simplex virus 1 infection, cytokine-cytokine receptor interaction, and NOD-like receptor signaling pathway. Thus, 21 hub genes were identified from PPI network, including IL6, IL1B, ITGAM, CXCL8, ITGAX, CCL5, SERPINA1, APOE, CSF1R, VWF, GBP1, APOB, CYBB, HLA-DRB1, CD68, FGG, FGA, OASL, NOD2, OAS2 and FCGR2A. These findings suggested that simulated microgravity upregulated inflammation-related genes and pathways of GES-1 cells, which may play important roles in the response to microgravity and provide useful information for preventing mucosal damage in astronauts. In conclusion, this study revealed the key genes and pathways associated with simulated microgravity and indicated that simulated microgravity induced an inflammatory response in gastric mucosal epithelial cells.