The present study aimed to investigate the key pathways and genes associated with gastric adenocarcinoma via transcriptome sequencing. Five pairs of gastric adenocarcinoma tissue and normal tumor‑adjacent tissue were harvested. After sequencing, raw data were processed and differentially expressed genes (DEGs) between tumor and control groups were screened, followed by functional enrichment analysis and gene clustering analysis. The effect of DEGs on patient prognosis was analyzed on the basis of the survival data from gastric adenocarcinoma patients in The Cancer Genome Atlas database. Several genes were validated through reverse transcription‑quantitative polymerase chain reaction. In total, 1,477 upregulated and 282 downregulated DEGs were screened in tumor groups. These genes were segregated into four clusters. Genes in cluster 1 were significantly involved in metabolism of xenobiotics by cytochrome P450, genes in cluster 2 were majorly involved in apoptosis, tight junction formation, and platelet activation, genes in cluster 3 were primarily enriched in the p53 signaling pathway and genes in cluster 4 were significantly enriched in the insulin resistance pathway. Furthermore, 15 DEGs significantly influenced prognosis, including F2R, CTHRC1, and RASGRP3. The expression levels of CYP2B6, MAPK13, CTHRC, RASGRP3 and PYGM were consistent with our analysis results. In conclusion, pathways for metabolism of xenobiotics via cytochrome P450, apoptosis, tight junction formation, platelet activation, and insulin resistance may serve important roles in the progression of gastric adenocarcinoma. Notably, CTHRC1 and RASGRP3 may serve as key prognostic markers.