Conscientious is associated with greater longevity and other favourable health outcomes, but the processes underlying these links are poorly understood. Health behaviours such as physical activity and avoidance of smoking and excessive alcohol consumption may contribute, but direct associations with neuroendocrine and inflammatory processes may also be relevant. We tested the associations between conscientiousness and hair cortisol concentration in 2318 older men and women (mean age 66.2 years) from the English Longitudinal Study of Ageing. Conscientiousness was positively associated with physical activity and fruit and vegetable consumption, and negatively related to alcohol intake, sedentary behaviour, body mass index and depressive symptoms (all p<0.001). We found an inverse association between conscientiousness and hair cortisol concentration that was independent of age, sex, education and wealth (β=-0.053, p=0.012), and the relationship remained significant with additional adjustment for health behaviour and depressive symptoms (β=-0.048, p=0.025). The observation that greater conscientiousness was correlated with lower hair cortisol indicates that this trait might impact central nervous regulation of hypothalamic-pituitary-adrenocortical function, with effects that are possibly advantageous for health.