In adipose tissue, glucocorticoids (GC) regulate lipogenesis and lipolysis. Hexose-6-phosphate dehydrogenase (H6PDH) is an enzyme located in the endoplasmic reticulum (ER) that provides co-factor for the enzyme 11-hydroxysteroid dehydrogenase type 1 (11-HSD1), regulating the set point of its activity and allowing for tissue specific activation of GCs. The aim of this study was to examine the adipose tissue biology of the H6PDH null (H6PDH/KO) mouse. Real-time PCR analysis confirmed similar mRNA levels of 11-HSD1 and glucocorticoid receptor (GR) in wild type (WT) and H6PDH/KO mice in liver and gonadal fat (GF) depots. Microsomal 11-HSD1 protein levels shown by Western Blot analysis corresponded well with mRNA expression in GF of WT and H6PDH/KO mice. Despite this, the enzyme directionality in these tissues changed from predominately oxo-reductase in WT to exclusively dehydrogenase activity in the H6PDH/KO mice. In the fed state, H6PDH/KO mice had reduced adipose tissue mass but histological examination revealed no difference in average adipocyte size between genotypes. mRNA expression levels of the key lipogenic enzymes; acetyl CoA carboxylase, adiponutrin and stearoyl-coenzyme A desaturase2 were decreased in H6PDH/KO mice, indicative of impaired lipogenesis. In addition, lipolysis rates were also impaired in the H6PDH/KO as determined by lack of mobilisation of fat and no change in serum free fatty acids (FFA) concentrations upon fasting. In conclusion, in the absence of H6PDH the set point of 11-HSD1 enzyme activity is ‘switched’ from predominately oxo-reductase to dehydrogenase activity in adipose tissue – as a consequence this leads to impairment of fat storage and mobilisation.