We examined whether a high cortisol awakening response (CAR) and low cortisol decline over the day (CDD) are related to self-reported work stress and well-being, and whether there are gender differences in these relationships. Three hundred eighty-three working men and women responded to a survey measuring job stress factors, mastery at work, symptoms and well-being. Salivary cortisol was sampled at awakening, after 45 min and at 21:00, from which the variables CAR and CDD were defined. A high CAR was associated with lower perceived job control and work mastery, and poorer well-being. Low CDD was associated only with higher job demands, but the self-report scores showed a number of interactions between cortisol group and gender. Among women, those showing a low CDD, compared with those with a higher CDD, had more favourable scores on a number of job stress factors and symptom load. In contrast, among men, a similar comparison showed those with low CDD to have poorer scores on job stress factors and symptom load. We conclude that individuals displaying high CAR or low CDD differ from those not displaying these cortisol profiles in self-report of work stress and well-being, and that gender differences appear in these relationships.