Purpose: Gestational diabetes mellitus (GDM) is one of the most common medical problems of pregnancy. This study is designed to identify serum biomarkers, which can predict the subsequent development of GDM at early stages. Experimental design: Maternal blood was obtained prospectively from pregnant women at 12-16 wk of pregnancy. Among these, 30 women were subsequently diagnosed with GDM at 24 to 28 wk and were selected as case studies along with 30 normoglycemic women as controls. Serum samples were analyzed by using iTRAQ analysis. Results: Thirty three differentially expressed proteins were identified between case and control groups. They were involved in various signaling processes previously implied in GDM. Of which four proteins, i.e. apolipoprotein E, coagulation factor IX, fibrinogen alpha chain, and insulin-like growth factor-binding protein 5 were successfully verified by ELISA. Combinations of these four proteins, the area under the receiver operating characteristic curve, sensitivity, and specificity were 0.985, 80% and 95%, respectively. Conclusion: The results highlight the roles of complement system, inflammatory and immune response, and blood coagulation in the pathogenesis of GDM. The panel of four candidate proteins could distinguish women subsequently developed with GDM from controls with high sensitivity and specificity.