Purpose. Glucocorticoid (GC) treatment impairs osteoblast function, undercarboxylated osteocalcin (ucOC), and insulin sensitivity. However, the effects of acute GC ingestion on post-exercise insulin sensitivity in humans are unclear. We investigated whether the suppression of ucOC, by a single dose of GC (prednisolone), would be associated with impaired post-exercise insulin sensitivity and skeletal muscle mTOR/insulin protein signalling. Methods. Nine healthy males (Age: 28 ± 2 years; BMI: 24 ± 1; Mean ± SEM) were randomly allocated in a double-blinded cross-over design to ingest a single dose of prednisolone (20 mg) and placebo, ~7 days between trials. Twelve hours after capsule ingestion, after an overnight fast, participants performed a session of high-intensity interval exercise (4 x 4-minute cycling intervals at 90-95% HRpeak, 2-minute active recovery periods). The homeostatic model assessment (HOMA2-IR) was used to assess resting insulin resistance and the euglycaemic-hyperinsulinaemic clamp (EHC) was used to assess insulin sensitivity 5 hours after exercise. Serum ucOC, and skeletal muscle AS160Thr642, AktSer473 and mTORSer2481 protein phosphorylation, were measured at baseline and post-EHC. Results. Compared to placebo, prednisolone treatment suppressed ucOC at baseline (-24±2%, p<0.001) and post-EHC (-18±2%), which coincided with increased HOMA2-IR (107±27%, p<0.001) and decreased post-exercise insulin sensitivity (-34±5%, p<0.001). Higher serum ucOC was associated with lower HOMA2-IR (r=-0.54, p<0.05) and greater post-exercise insulin sensitivity (r=0.72, p<0.01). Prednisolone significantly impaired (p<0.05) the post-exercise insulin stimulated increase in skeletal muscle AS160Thr642 (~-50%), AktSer473 (~-61%) and mTORSer2481 (~-59%) phosphorylation, which significantly correlated (p<0.01) with lower serum ucOC (r=0.64, r=0.71 and r=0.61, respectively) and post-exercise insulin sensitivity (r=0.56, r=0.75, r=0.54, respectively). Conclusions. The negative effect of prednisolone on insulin sensitivity at rest and following exercise are related, at least in part, to the suppression of ucOC and mTOR/insulin signalling. Targeting ucOC mediated signalling pathways in humans may prove to be an effective intervention for improving glycaemic control in insulin resistant populations.