The role of endogenous glucocorticosteroids (GC) in bone development is ill-defined. Using the Col2.3-11βHSD2 transgenic (tg) mouse model, we examined the effect of osteoblast-targeted disruption of intracellular GC signalling on bone growth and strength, and its modulation by factors such as age, gender and skeletal site. Tibiae and L3 vertebrae of 3 and 7-week-old, male and female wild type (WT) mice and their tg, age and sex matched littermates were analysed by micro-CT and mechanical testing. Data were analysed separately for 3 and 7-week-old mice by 2-way ANOVA using genotype (WT, tg), gender and their interactions as factors. Transgenic mice were characterised by lower bone volume, lower trabecular number and higher trabecular separation in tibial trabecular bone, as well as lower tibial cortical bone area and periosteal and endosteal perimeters. These changes resulted in a marked decrease in mechanical bone strength and stiffness in sexually mature, 7-week-old mice. In the tibia, the observed transgene effect was present in 3 and 7-week-old animals, indicating that the biological effect of disrupted GC signalling was independent of sexual maturity. This was not the case for the vertebral bones, where significant differences between tg and WT mice were seen in 7 but not in 3-week-old animals, suggesting that the effects of the transgene at this site may be modulated by age and/or changes in circulating sex hormone levels. Taken together, our results demonstrate that endogenous glucocorticoids may be required for normal bone growth but that their effect on bone structure and strength varies according to the skeletal site and sexual maturity of the animals.