Large-scale studies on genetic risk loci for melatonin receptor 1B (MTNR1B) gene and GDM risk have not been well generalized to the Chinese population. In this study, we performed two-stage case–control study: 1.429 pregnant women: 753 GDM/676 controls in the Southern Chinese population by genotyping 5 SNPs (rs10830963, rs1387153, rs2166706, rs1447352, and rs4753426) in MTNR1B. Genotypes were determined using the Sequenom MassARRAY platform and TaqMan allelic discrimination assay. Interactions between genetic variants and age/BMI as predictors of GDM risk were evaluated under the logistic regression model. In the first stage, the SNP rs10830963 was discovered to be potentially related to GDM risk (additive model: OR = 1.27, 95%CI = 1.05–1.55, P = 0.025), which was further confirmed in the second stage with a similar effect (additive model: OR = 1.53, 95%CI = 1.19–1.98, P = 0.005). In the combined stage, the G allele of rs10830963 was potentially associated with GDM risk (additive model: OR = 1.36, 95%CI = 1.17–1.59, P < 0.001; dominant model: OR = 1.45, 95%CI = 1.15–1.83, P = 0.005). The rs10830963 interacted with age and BMI to contribute to GDM risk in the combined participants. And, the similar interactive effects for the other four SNPs also exist. These findings offer the potential to improve our understanding of the etiology of GDM, and particularly of biological mechanisms.