Objective Loneliness in older adults has been associated with increased mortality and health problems. One of the assumed underlying mechanisms is dysregulation of the hypothalamic-pituitary-adrenocortical axis (HPA–axis). The purpose of this study was to investigate whether loneliness in older adults is associated with HPA-axis dysregulation and whether this association differs between depressed and non-depressed persons. Methods Cross-sectional data of 426 lonely and non-lonely older adults in the Netherlands Study of Depression in Older Persons (NESDO) were used. Linear regression analyses and multinominal logistic regression analyses were performed to examine the association between loneliness and morning cortisol, cortisol awakening response, diurnal slope and dexamethasone suppression ratio. In all analyses, confounders were introduced. In order to examine whether the association between loneliness and cortisol measures is different in depressed versus non-depressed persons, an interaction term for loneliness x depression diagnosis was tested. Results Cortisol output in the first hour after awakening and dexamethasone suppression ratio was lower in lonely participants. There were no significant interactions between loneliness and depression diagnosis in the association with the cortisol measures. Conclusion This study is the first to investigate the association between the HPA–axis and loneliness in a large group of older adults aged 60–93 years. We found lower cortisol output in the first hour after awakening and lower dexamethasone suppression ratio in lonely older depressed and non–depressed adults. Whether diminished cortisol output is the underlying mechanism that leads to health problems in lonely older adults is an interesting object for further study.