Galectin-9 (Gal-9), a member of the β-galactoside-binding galectin family, plays a role in immune response, apoptosis, cell proliferation and cell death. Recent studies have shown that abnormal expression of Gal-9 is involved in certain primary cancers. The present study is the first investigation of the role of Gal-9 gene expression in clinically diagnosed primary gastric cancer tissues. Gal-9 mRNA expression was assessed in 44 clinically diagnosed frozen primary cancer tissue samples using quantitative PCR (qPCR). Analysis of the qPCR data revealed a significant reduction (>2-fold decreased) of Gal-9 gene expression in gastric cancer tissues in 77% (34/44) of patients. In patients with gastric cancer, although no statistically significant difference was found between adjacent (<2 cm away from the cancer tissue) and normal tissues (>5 cm away from the cancer tissue), a >2-fold reduction in Gal-9 expression was observed in the adjacent tissues of 34% of the patients. Compared to matched normal or adjacent tissues, the gene expression of Gal-9 was significantly decreased in tumor tissues (p<0.001). The correlation of Gal-9 expression and clinicopathological features in gastric cancer was analyzed according to the TNM classification system using AJCC stage grouping. In patients with gastric cancer, clinical staging, tumor pathological stage (pT stage), tumor cell differentiation, lymph node metastasis and survival rate were found to be associated with Gal-9 expression. However, no significant association was found between Gal-9 expression and distant metastasis (p>0.05). No significant difference was found between patients of different genders, levels of cell differentiation, distant metastasis status or different survival time of patients. Compared to normal tissues, >2-fold reduction of Tim-3 expression in gastric cancer tissues occurred in 59% of patients, but no correlation was found between Gal-9 and Tim-3 in gastric cancer. These results strongly suggest that Gal-9 is involved in tumorigenesis of gastric cancer.