Background Hypothalamic-pituitary-adrenal (HPA) axis adaptation is a potential mechanism linking early life exposures with later adverse health. This study tested the hypothesis that preterm birth is associated with adaptation of diurnal cortisol regulation across infancy. Methods A secondary analysis was conducted of saliva cortisol measured morning, midday and evening, monthly, across infancy, as part of a birth cohort conducted in Linkoping, Sweden. Diurnal cortisol regulation of infants born extremely preterm (n=24), very preterm (n=27) and at term (n=130) were compared across infancy through random coefficients regression models. Results Compared with infants born at term, infants born extremely preterm (-17.2%, 95% CI: -30.7 to -1.2), but not very preterm (1.7%, 95% CI: -14.1 to 20.4), had a flattened diurnal slope across infancy. Conclusions Extremely preterm birth is associated with a flattened diurnal slope in infancy. This pattern of cortisol regulation could contribute to adverse metabolic and neurodevelopmental phenotypes observed in this population.