Single-nucleotide polymorphisms (SNPs) of microRNAs (miRNAs) may alter miRNA expression, binding affinity, and/or messenger RNA expression levels of the target genes, thus leading to disease susceptibility. This study explored the association between SNPs in neuroendocrine stress response-related miRNAs and type 2 diabetes (T2D). In the screening stage, the association between six candidate SNPs of miRNAs and T2D was analyzed in a case-control study including 504 T2D cases and 494 healthy controls. Homozygous GG genotype of pri-miR-144 rs9279 showed significant association with increased risk of T2D compared with homozygous TT genotype (adjusted odds ratio [OR] = 1.62, 95% confidence interval [CI]: 1.07-2.45;p = .023) and the combined TT/TG genotype (adjusted OR = 1.59, 95% CI: 1.08-2.36;p = .020). In the validation stage, the association was further validated in a second independent set of subjects. The GG genotype showed consistent directions and effect sizes that were identified in previous additive and recessive models. The expression levels of miRNAs were further compared between different genotypes in the 179 newly diagnosed cases and 183 frequency-matched healthy controls. As a result, the GG genotype carriers had significantly upregulated expression of plasma miR-144 and cortisol, as compared to individuals with TT and TG genotypes, respectively, in total subjects and subgroups (p < .05). Eventually, NR3C1 was proved to be a stress-related target gene of miR-144, indicating that pri-miR-144 rs9279 may contribute to the development of T2D by altering regulation of stress response.