Accumulating evidence shows that runt-related transcription factor 3 (Runx3) is a putative tumor suppressor in various types of cancer, the lower levels of which are associated with a less favorable cancer outcome. However, these studies were restricted to primary cancer lesions. Lymph node metastasis (LNM) is a significant factor in determining the prognosis of patients with gastric cancer and is a frequent target of chemotherapy. In the present study, we investigated the expression of Runx3 in the lymph nodes (LNs) of stomach carcinoma and the association of Runx3 expression with the prognosis of patients. The expression of Runx3 in LNs with and without metastasis was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The positive rate of Runx3 mRNA in LNM specimens was significantly lower (28.4%, 21 out of 74) compared to that of the non-metastatic samples (33.3%, 9 out of 27, P<0.05). Similar findings were obtained by Western blotting. Univariate analysis revealed that the loss of Runx3 expression in LNs was not only associated with poor clinicopathological factors, such as LNM, distant organ metastasis, later clinicopathological stages and deep infiltration, but also with a lower 5-year survival rate and poorer prognosis. These results strongly suggest a potential diagnostic value of Runx3 expression in LNs and multiple pathways contributing to the outcome of patients with gastric cancer.