Emerging evidence suggested genetic factor attributed as a major determinant for the complex pathogenic mechanism of gestational diabetes mellitus (GDM), but the related genetic study was limited. We aimed to investigate the impact of polymorphisms in miRNA-binding sites (miR-binding SNPs) on the risk of GDM in Chinese Han pregnant women. We screened GDM susceptibility genes extensively and selected miR-binding SNPs using four bioinformatics software. TaqMan allelic discrimination assays were applied to miR-binding SNPs genotyping in 839 GDM patients and 900 controls. In total five potential miR-binding SNPs (SLC30A8 rs2466293, INSR rs1366600, INSR rs3745550, KCNJ11 rs5210 and KCNQ1 rs8234) were selected. Our results showed that SLC30A8 rs2466293 [OR 95% CI = 1.455 (1.077, 1.966); P = 0.014] and INSR rs1366600 [OR 95% CI = 2.191 (1.077, 4.455); P = 0.029] increased the risk of GDM after adjusting age in additive model. Furthermore, rs2466293 was found to significantly associate with higher levels of fasting plasma glucose (b (dom) = 0.054, P (dom) = 0.032), 2-h OGTT plasma glucose (b (dom) = 0.069, P (dom) = 0.007), lower fasting insulin concentrations (b (rec) = -0.082, P (rec) = 0.003) and decreased HOMA-B (b (rec) = -0.067, P (rec) = 0.015). Additionally, the correlation between rs1366600 and 2-h OGTT plasma glucose (b (dom) = 0.078, P (dom) = 0.001) was observed. Two miR-binding SNPs SLC30A8 rs2466293 and INSR rs1366600 increased GDM susceptibility. Functional studies were required to confirm the underlying mechanism. Our study provided additional insights into the genetic pathogenesis of GDM.