Functional cross-talk between the hypothalamic-pituitary-adrenal and -gonadal (HPA and HPG) axes is subject to metabolic influence in basal and stress conditions. However, how testicular steroids attributed to the homeostatic steroidogenesis in the absence of adrenal glands remains unclear. The GC-MS/MS-based quantitative profiling of 57 steroids were, therefore, applied into 8-week-old male C57BL/6 mice with bilateral adrenalectomy (bADX), which were subdivided by tap-water (n=20) and saline (n=37) groups, and sham controls (n=16). Testes samples were taken at 12 weeks after bADX and 21 testicular steroids were quantitatively compared between groups. Decreased corticosterone levels in testes were found in both bADX groups compared with those in the controls (p < 0.005), while no significant changes in other steroids were observed. Tetrahydro-11-deoxycorticosterone (THDOC) showed the lower levels in saline group than tap-water counterpart (p < 0.03). In contrast to most of metabolic ratios, which were decreased in bADX mouse testes, a metabolic ratio of testosterone to androstenedione was increased in both tap-water and saline groups (p < 0.05 and p = 0.054, respectively) than in the control group. Increased testosterone and decreased corticosterone in testes of bADX models might be associated with cross-talk of HPA and HPG axes, and THDOC could be indicative for hypertensive stress in testes.