Glucocorticoid concentrations are a balance between production under the negative feedback control and diurnal rhythm of the hypothalamic-pituitary-adrenal (HPA) axis and peripheral metabolism, for example by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which catalyses the reduction of inactive cortisone (11-dehydrocorticosterone (11-DHC) in mice) to cortisol (corticosterone in mice). Reductase activity is conferred upon 11β-HSD1 by hexose-6-phosphate dehydrogenase (H6PDH). 11β-HSD1 is implicated in the development of obesity, and selective 11β-HSD1 inhibitors are currently under development. We sought to address the concern regarding potential up-regulation of the HPA axis associated with inhibition of 11β-HSD1. We assessed biomarkers for allele combinations of 11β-HSD1 and H6PDH derived from double heterozygous mouse crosses. H6PDH knock out (KO) adrenals were 69% larger than WT while 11β-HSD1 KO and double KO (DKO) adrenals were ~30% larger than WT - indicative of increased HPA axis drive in KO animals. ACTH-stimulated circulating corticosterone concentrations were 2.2-fold higher in H6PDH KO animals and ~1.5-fold higher in 11β-HSD1 KO and DKO animals compared with WT, proportional to the observed adrenal hypertrophy. KO of H6PDH resulted in a substantial increase in urinary DHC metabolites in males (65%) and females (61%). KO of 11β-HSD1 alone or in combination with H6PDH led to significant increases (36 and 42% respectively) in urinary DHC metabolites in females only. Intermediate 11β-HSD1/H6PDH heterozygotes maintained a normal HPA axis. Urinary steroid metabolite profile by gas chromatography/mass spectrometry as a biomarker assay may be beneficial in assaying HPA axis status clinically in cases of congenital and acquired 11β-HSD1/H6PDH deficiency.