Aims: Serum cystatinC (CysC) has recently been shown to be associated with the incidence of type 2 diabetes mellitus (T2DM) and progression to the pre-diabetic state. The aim of this study was to explore the relationship between serum CysC and the risk of gestational diabetes mellitus (GDM) in Chinese pregnant women. Methods: This cross-sectional study consisted of 400 pregnant women including 111 with GDM and 289 with normal glucose tolerance at 24-28 weeks of gestation. The subjects were further divided into four groups according to the CysC quartiles, and their clinical characteristics were compared. The serum CysC concentration was measured using immunoturbidimetry and the degree of insulin resistance was assessed by the homeostasis model assessment of insulin resistance (HOMA-IR). Results: Serum CysC levels were significantly higher in pregnant women with GDM than in the healthy pregnant women [1.0(0.8-1.8) vs 0.7(0.6-1.0), P<0.01). The Spearman's correlation analysis showed that serum CysC was positively associated with HOMA-IR(r=0.118, P<0.05) and the occurrence of GDM (r=0.348, P<0.01). The pregnant women were divided into quartiles according to their serum CysC concentrations. Compared to the first quartile, pregnant women in Q2 (OR, 2.441; P=0.025), Q3 (OR, 3.383; P=0.001) and Q4 (OR, 5.516; P<0.001) had higher risk of GDM after adjusted for age, BMI, HbA1c and HOMA-IR. Further, with a rise in the serum CysC, there was an increasing trend in the HOMA-IR levels (P<0.05). A binary logistic regression analysis after adjusting for other confounding variables revealed a significant and independent association between serum CysC and GDM [OR=14.269; 95% confidence interval, 4.977-40.908, P<0.01]. The receiver operating characteristic curve analysis revealed that the optimal cutoff point for serum CysC to indicate GDM was 0.95mg/L. Conclusions: Serum CysC is significantly and independently associated with insulin resistance and GDM. It may be a helpful biomarker to identify the risk of GDM in Chinese pregnant women.