Objective: Emerging evidence has suggested that glycemic variability plays an important role in the development of diabetes complications. We aimed to evaluate the influence of c-peptide levels on glycemic variability and hypoglycemia in patients with GAD-antibody negative, insulin-treated type 2 diabetes. Methods: We enrolled 80 patients with insulin-treated type 2 diabetes (mean age 62.6 ± 9.4 years, mean HbA1C 71.9 ± 11.9 mmol/mol, mean BMI 33.1 ± 5.7 kg/m2). All patients were GAD-antibody negative, had long diabetes duration (18.9 ± 7.4 years) and were treated with basal-bolus insulin. Glycemic variability and hypoglycemia duration were assessed from continuous glucose monitoring data recorded over 6 consecutive days. Glycemic variability was assessed by calculating mean Coefficient of Variation (CV). Hypoglycemia was defined as BG ≤70 mg/dl (3.9 mmol/L) or BG <54 mg/dl (3.0 mmol/L). Fasting c-peptide and fasting glucose were measured on day 1. Results: Mean fasting c-peptide was 647 ± 493 pmol/L. Low levels of fasting c-peptide were correlated to higher CV (r = -0.44, P=0.001). In a multivariate regression model with HbA1C, BMI, diabetes duration and total daily insulin dose only c-peptide was significantly associated with CV. 47,5% of the patients had at least one episode of hypoglycemia (BG ≤70 mg/dl) and 23.7% had at least one episode of BG <54 mg/dl. Patients with at least one episode of hypoglycemia had significant lower c-peptide than patients without hypoglycemia (494 ± 460 pmol/L vs. 821 ± 479 pmol/L, P=0.004). There was no significant difference in c-peptide between patients with BG ≤70mg/dl and patients with BG <54mg/dl. Conclusion: Low levels of c-peptide are associated with higher glycemic variability and risk of hypoglycemia in GAD-antibody negative patients with insulin treated type 2 diabetes, suggesting a role for c-peptide in optimizing treatment and prevention of hypoglycemia in insulin-treated type 2 diabetes.