Objective: To investigate the expressions of miR-23a and runt-related transcription factor 1 (RUNX1) in gastric cancer and their significances. Methods: The binding affinity between the miR-23a and 3' untranslated region (3'UTR) of RUNX1 was evaluated by luciferase reporter gene activity assay. The resection specimens (gastric cancer tissues and their adjacent tissues) from 87 gastric cancer patients were collected, and then the expressions of miR-23a and RUNX1 were detected by in situ hybridization and immunohistochemical staining, respectively. Results: The uciferase reporter gene activity assay suggested that RUNX1 was the target gene of miR-23a. The positive expression rates of miR-23a and RUNX1 in the 87 cases of gastric cancer tissues were 82.76% and 14.94% respectively, and both were significantly higher than those (25.29% and 80.46%) in the adjacent tissues (both P<0.01). The expression of miR-23a in the cases with lymph node metastasis was significantly higher than that in the cases without lymph node involvement (P<0.01), and the miR-23a expression increased with the development of the clinical stage and infiltration depth (P<0.01). The expression of RUNX1 in the cases with lymph node metastasis was significantly lower than that those without lymph node involvement (P<0.01), and the RUNX1 expression decreased with the development of the clinical stage and infiltration depth (P<0.01). There was a significant negative correlation between the expression of miR-23a and RUNX1 in gastric cancer (r=–0.474, P=0.004). Conclusion: RUNX1 is the target gene directly regulated by miR-23a. The high miR-23a expression and lower RUNX1 protein expression may be important biological markers for malignant transformation of the gastric mucosa, and invasion or metastasis of gastric cancer.