Background: The aim of the study was to evaluate the relationship between serum Sestrin2, Insulin resistance (IR), metabolic syndrome (MetS), and diabetic nephropathy (DN) in patients with type 2 diabetes mellitus (T2DM). Patients and methods: The study group consisted of 155 patients with T2DM who were recruited and evaluated for DN. IR was evaluated by the homeostasis model assessment-insulin resistance (HOMA-IR). Sestrin2 levels were determined by enzyme-linked immunosorbent assay. Results: Serum Sestrin2 levels showed significant positive correlations with tumor necrosis factor-α (r=0.247, P=0.018), triglycerides (r=0.178, P=0.021), total cholesterol (r=0.33, P=0.007), fasting c-peptide (r=0.164, P=0.035), and systolic blood pressure (r=0.171, P=0.041). A significant negative correlation was found between Sestrin2 levels and serum adiponectin (r=0.247, P=0.018). Also, Sestrin2 levels were positively correlated HOMA-IR (r=0.188, P=0.026). There was a progressive significant increase of mean HOMA-IR through Sestrin2 tertiles (P=0.001). A significant increase of mean serum Sestrin2 levels was found in relation to the presence of DN or MetS. Conclusion: Serum Sestrin2 levels are significantly associated with IR, MetS, and DN in patients with T2DM.