MicroRNA plays an important role in spermatogenesis. Whether pre-miRNAs polymorphisms are associated with idiopathic male infertility remains obscure. In this study, 1378 idiopathic infertile males and 486 fertile controls were included between 2006 and 2014. Genotype of three polymorphisms (hsa-mir-146a rs2910164, hsa-mir-196a-2 rs11614913, and hsa-mir-499 rs3746444) and expression of miRNA in seminal plasma were examined by TaqMan method. The role of hsa-miR-196a-5p in cell proliferation, apoptosis and cell cycle were also examined in GC-2 cells. Our results demonstrated that rs11614913 of hsa-miR-196a-2 was significantly associated with idiopathic infertility (TT vs. CT: P = 0.014; TT vs. CC: P = 0.005; TT vs. CT + CC: P = 0.003). In following stratified analysis, we found that rs11614913 exhibited a significantly higher risk of asthenospermia, oligozoospermia and azoospermia. However, no significant association was observed between the other two polymorphisms and idiopathic male infertility risk. In a genotype-expression correlation analysis, rs11614913 CC was significantly associated with elevated expression of hsa-miR-196a-5p (P < 0.05). Additionally, apoptosis levels were significantly increased in hsa-miR-196a-5p mimic treated GC-2 cells, while decreased in hsa-miR-196a-5p inhibitor treated GC-2 cells. Our data revealed a significant relationship between hsa-miR-196a-2 polymorphism and idiopathic male infertility.