Objective Stress is an important component in the pathophysiology of irritable bowel syndrome (IBS). Long term Hypothalamus Pituitary Adrenal (HPA)-axis activity can be studied by measuring hair cortisol concentrations (HCC). Some previous studies have indicated a dysregulated HPA-axis in IBS patients, but cortisol levels in hair have not yet been studied. We investigated whether HCC and self-reported stress differentiate IBS patients from controls. Methods In a cross-sectional study within 10 Swedish Primary Health Care Centers we compared patients in working age with active IBS to patients without GI complaints. The participants donated hair samples and completed questionnaires including a scale of self-reported perceived stress (PSS). 169 Rome III-fulfilling IBS patients and 316 non-IBS patients were available for final analyses. Results IBS patients had significantly lower HCC, median = 16.3 pg/mg, IQR = 26.9 pg/mg, compared to non-IBS patients, median = 22.8 pg/mg, IQR = 29.1 pg/mg. There was also a difference in the distribution of HCC quintiles between the two groups, with 30.2% IBS patients and 14.2% of non-IBS patients in the lowest quintile of HCC. PSS was higher among IBS patients with a mean (SD) total score of 25.3 (8.0) compared to controls 21.4, (7.5). Quintiles of HCC and PSS stayed significantly but very weakly related to IBS (B = − 0.332, Std error = 0.146, p < 0.005) in multivariable analyses. Conclusion This study suggests a possible suppression of the HPA-axis activity in a considerable portion of IBS patients.