Background: Hyperglycaemia is a common complication of prematurity and is associated with neonatal mortality and morbidity, yet the aetiology is incompletely understood. C-peptide has been used in adults to estimate endogenous insulin secretion due to its simple clearance kinetics. Objective: To determine insulin secretion calculated from plasma C-peptide concentrations in hyperglycaemic preterm babies. Methods: A retrospective analysis of a cohort of 41 very preterm babies (median gestational age, weeks: 27.2 (26.2-28.7)) enrolled in a randomised controlled trial of tight glycaemic control when they developed hyperglycaemia (2 consecutive blood glucose concentrations (BGC) > 8.5 mmol.L-1). Insulin secretion was determined using a steady state analysis of a 2 compartment C-peptide kinetic model. Results: BGC, plasma insulin concentration, plasma C-peptide concentrations and insulin secretion were higher at randomisation than 1-2 weeks following randomisation (p≤0.02). Insulin secretion was higher in girls (11.7 (5.3-18.7) vs. 4.7 (2.1-8.3) mU.L-1.kg-1.min-1, p<0.005) with no difference in clinical characteristics, BGC, plasma insulin concentration, or nutrition between the sexes (p>0.25). Insulin secretion was lower in samples taken during exogenous insulin delivery (3.7 (1.8 - 6.9) vs. 9.8 (4.7 - 17.8) mU.L-1.kg-1.min-1, p=0.02). Conclusions: Insulin secretion was higher when babies had higher BGC, indicating endogenous insulin secretion is sensitive to BGC. Girls had higher insulin secretion, at similar blood glucose and plasma insulin concentrations, than boys.