Objective <p>To investigate the expression levels of high mobility group box 1 (HMGB1) and nuclear factor kappa-B (NF-κB) and to characterize their relationship in gastric adenocarcinoma.</p> Methods <p>The expression data for HMGB1 and NF-κB from the GEO database were analyzed to assess expression levels and prognostic relevance in gastric cancer. Immunohistochemistry was performed to verify their expressions in primary gastric adenocarcinoma tissues and adjacent non-tumor tissues. Next, we collected clinicopathologic data of patients with gastric cancer and analyzed the correlations between HMGB1 and NF-κB expression. Furthermore, we assessed the impact of HMGB1 and NF-κB expression on the survival time of gastric cancer patients using Kaplan–Meier survival analysis. All statistical analyses were conducted using GraphPad Prism 9.</p> Result <p>The results showed that HMGB1 and NF-κB were significantly higher than normal tissues, which were significantly correlated with survival prognosis. IHC analysis further confirmed high positivity rates for HMGB1 (81.76%) and NF-κB (75%) in gastric adenocarcinoma, respectively. Furthermore, higher expression levels of HMGB1 and NF-κB were associated with shorter survival time of gastric cancer patients. The expression level of HMGB1 or NF-κB was correlated with tumor size, depth of invasion, and clinical stage. Finally, we demonstrated that there is a correlation between the expression of HMGB1 and NF-κB.</p> Conclusion <p>Previous studies focused on mechanistic regulation, while systematic clinicopathologic co-expression analysis of HMGB1 and NF-κB in human gastric adenocarcinoma tissues remains limited. This work provides novel evidence that the co-expression of HMGB1 and NF-κB is associated with key clinicopathologic factors and survival outcomes in gastric adenocarcinoma.</p>