Objective To investigate the viability of Runt-related transcription factor 1 (RUNX1) as a biomarker for gastric cancer and to assess the impact of the small molecule inhibitor Ro24-7429 on the proliferation, migration, and invasion of gastric cancer cells following targeted modulation. Methods Through the GEPIA database, we analyzed RUNX1 mRNA expression in gastric cancer or normal gastric tissues. Utilizing RUNX1 expression data from the TCGA database, a receiver operating characteristic (ROC) curve was constructed to appraise the potential of RUNX1 as a gastric cancer biomarker. In September 2022, we collected tissue samples from 6 patients with gastric cancer from the Department of General Surgery at the Second Hospital of Lanzhou University. After extracting tissue proteins, Western blotting was employed to compare RUNX1 protein expression in tumor and adjacent tissues. Gastric cancer cell lines with high RUNX1 expression were identified and the suppressive effect of the small molecule inhibitor Ro24-7429 on RUNX1 protein expression was verified by Western blotting. the effect of Ro24-7429 was validated by using CCK-8, colony formation, cell scratch, and Transwell assays. RUNX1 protein levels in gastric cancer tissues were quantified using immunohistochemical staining. An organoid model of gastric cancer was then established from the high-expression samples and verified by both HE and immunization analyses. Lastly, the impact of Ro24-7429 on the growth of gastric cancer organoids with meticulous tracking was evaluated using a biological microscope within a designated area. Results The analysis from the GEPIA database revealed a heightened expression of RUNX1 mRNA in gastric cancer tissues compared with normal tissues (P<0.05). The ROC curve derived from the RUNX1 expression data in the TCGA database boasts an area under the curve (AUC) of 0.956, underscoring RUNX1's potential as a robust diagnostic marker. Western blotting results revealed significantly higher RUNX1 protein expression in gastric cancer tissues than in adjacent tissues (P<0.001). Among 5 gastric cancer cell lines studied, AGS and HGC27 exhibited pronounced RUNX1 protein expression (P<0.001). The small molecule inhibitor Ro24-7429, targeting RUNX1, potently suppressed RUNX1 expression in gastric cancer cells. The results from CCK-8, colony formation, scratch, and Transwell assays showed that Ro24-7429 effectively inhibited proliferation, migration, and invasion of gastric cancer cells (P<0.001). In a gastric cancer organoid model derived from high RUNX1 expression samples, the RUNX1 expression was remarkably consistent with its originating tissue. As expected, upon the targeted inhibition of RUNX1 using Ro24-7429, the cancer organoids significantly reduced growth capacity. Conclusions RUNX1 shows potential as a biomarker for gastric cancer. Ro24-7429 specifically inhibits RUNX1 expression and suppresses tumor cell proliferation, migration, and invasion in gastric cancer cell lines and organoid models.