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Objective The present study investigates the influence of genetic variants in miRNA machinery genes (DROSHA, DICER, AGO1, and GEMIN4) on gastric cancer in Chinese Han population, further revealing the genetic mechanisms of gastric cancer occurrence and development. Methods Genotyping of single nucleotide polymorphisms (SNPs) was performed in 628 patients with GC and 502 frequency-matched (age and gender) controls by the high resolution melting (HRM) method. Results The SNPs rs3742330 (DICER) and rs7813 (GEMIN4) were associated with susceptibility to gastric cancer (P = 0.002 and 0.010, respectively). Stratified analysis showed that the G allele of rs3742330 and genotype TT as well as T allele of rs7813 were associated with a later stage of gastric cancer (P=0.027, 0.032 and 0.018, respectively). Furthermore, the genotype TT and T allele of rs7813 appeared to be associated with a higher level of lymphatic metastasis of gastric cancer (P=0.021 and 0.030, respectively), while the genotype AA and A allele of rs636832 (AGO1) were correlated with a lower level of lymphatic metastasis of gastric cancer (P=0.016 and 0.041, respectively). There was no significant association between rs10719 (DROSHA) and gastric cancer. Conclusion The present data demonstrated that genetic variants in miRNA machinery genes had a significant association with GC susceptibility (DICER and GEMIN4) and malignant behavior such as tumor stage (DICER and GEMIN4) and lymphatic metastasis of GC (GEMIN4 and AGO1) in Chinese Han population.