Background: Increased serum cystatin C (CysC) can predict the onset of type 2 diabetes (T2D). Meanwhile, impaired pancreatic α- and β-cell functions get involved in the pathophysiological processes of T2D. So this study was to explore the relationships between serum CysC levels and pancreatic α- and β-cell functions in T2D. Methods: In this cross-sectional observational study, total 2634 patients with T2D were consecutively recruited. Each recruited patient was received serum CysC test and oral glucose tolerance test for synchronous detection of serum C-peptide and plasma glucagon. As components of pancreatic β-cell function, insulin secretion and sensitivity indices were evaluated by C-peptide area under curve(AUC-CP) and C-peptide-substituted Matsuda’s index(Matsuda-CP), respectively. Fasting glucagon(F-GLA) and post-challenge glucagon calculated by glucagon area under curve(AUC-GLA) were used to assess pancreatic α-cell function. These indices were skewed and were further natural log-transformed(ln). Results: With quartiles of serum CysC levels ascending, AUC-CP, F-GLA and AUC-GLA were increased, while Matsuda-CP was decreased(p for trend<0.001). Moreover, serum CysC levels were positively related to lnAUC-CP, lnF-GLA and lnAUC-GLA (r=0.241, 0.131 and 0.208, respectively, p<0.001), and inversely related to lnMatsuda-CP(r= –0.195, p<0.001). Furthermore, after controlling for other relevant variables via multivariable linear regression analysis, serum CysC levels were identified to account for lnAUC-CP (β=0.178, t=10.518, p<0.001), lnMatsuda-CP (β= –0.137, t= –7.118, p<0.001), lnF-GLA (β=0.049, t=2.263, p=0.024) and lnAUC-GLA (β=0.121, t=5.730, p<0.001). Conclusions: Increased serum CysC levels may be partly responsible for increased insulin secretion from β-cells, decreased systemic insulin sensitivity, and elevated fasting and postprandial glucagon secretion from α-cells in T2D.