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Objective This study was carried out to investigate the role of sex in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and its relationship with testosterone levels in male and female obesity. Research methods and procedures Twenty-two obese men (OB-M) and 29 obese women (OB-W) participated in the study. Two groups of normal weight men (NW-M) and women (NW-W), respectively, served as controls. In basal conditions, blood concentrations of major androgens, sex hormone-binding protein, and gonadotropins were assessed, and the free androgen index (testosterone x100/sex hormone-binding globulin) was calculated. All subjects underwent a combined corticotropin-releasing hormone plus arginine-vasopressin stimulation test. Results OB-M and NW-M had higher basal adrenal cortical tropic hormone (ACTH) and cortisol levels than their female counterparts. In addition, ACTH, but not cortisol basal, levels were significantly higher in obese than in normal weight controls in both sexes. OB-W had a higher response than OB-M to the combined corticotropin-releasing hormone plus arginine-vasopressin test of both ACTH and cortisol [expressed as incremental percentage of area under the curve (AUC%)]. The same finding was present between NW-W and NW-M. Basal luteinizing hormone levels were negatively correlated to ACTH(AUC%) in both OB-W and OB-M. In the OB-W, however, a positive correlation was found between cortisol(AUC%) and testosterone (r = 0.48; p = 0.002), whereas a tendency toward a negative correlation was present in OB-M. Discussion In conclusion, we have shown a significant positive relationship between the activity of the HPA axis and testosterone in obese women, which suggests a partial responsibility of increased HPA axis activity in determining testosterone levels. In addition, it clearly seems that, as reported in normal weight subjects, a sex difference in the HPA axis activity still persists even in the presence of obesity.