Introduction: Gestational diabetes mellitus (GDM) occurs in ∼10–25% of pregnancies. Nesfatin-1, plays a role in carbohydrate metabolism by inhibiting glucagon secretion, besides has a glucose-dependent insulinotropic effect. Explanation of the GDM pathogenesis is important due to preventing gestational complications. We aimed to investigate relationship between GDM and Nesfatin-1. Material and methods: Seventy-nine pregnant subjects were randomly allocated to either GDM group (GDG, n = 38) or control group (CG, n = 41). For GDM diagnosis, 50 and 100 g oral glucose tolerance test (OGTT) were used. Nesfatin-1, insulin and other parameters were measured for all subjects. The homeostasis model assessment-insulin resistance (HOMA-IR) was calculated. Results: Nesfatin-1 was found lower and insulin was found higher in GDG than CG. Negative correlation has been founded between Nesfatin-1 with weight, BMI, fasting glucose, serum glucose level at first hour of the 50 g OGTT and HOMA-IR. Conclusion: In this study, patients with GDM had lower Nesfatin-1 levels than without GDM. Therefore, when the Nesfatin-1 effects on the GDM pathogenesis is clear, it may be contributed to diagnosis and treatment of the GDM.