Single-nucleotide polymorphisms in microRNAs are related to the occurrence, development and prognosis of cancer. The aim of the present study is to investigate the possible influence of the miR-499(rs3746444) polymorphism on the risk of gastric cancer in Chinese population. A total of 363 GC patients and 969 cancer-free controls were enrolled in this study. The genotypes were obtained using MassARRAY method. The results showed that, compared with T allele, C allele was associated with a significantly increased risk of GC (OR = 1.491, 95% CI = 1.155-1.923, P = 0.002). Moreover, a significantly increased risk of GC in subjects with the TC genotype was observed (adjusted OR of 1.559, 95% CI = 1.148-2.117, P = 0.004), compared with the wide type TT. We also found that basically dominant model (TT vs. TC + CC) was suitable for the association between rs6513497 and the risk of GC (OR = 1.568, 95% CI = 1.173-2.097, P = 0.002). However, the same association was also shown in males and females. Meanwhile, rs3746444 was associated with the tumor size of GC patients. The present study indicated that miR-499 rs3746444 might contribute to GC risk and this SNP could be developed as a biomarker for GC prediction.