The aim of this study was to investigate the acute effects of oral glucocorticoids in doses used in clinical practice on biochemical indices of the function of osteoclasts, osteoblasts, and osteocytes. In 17 adult patients suffering from various medical pathologies requiring systemic steroid therapy that were never before treated with glucocorticoids, glucocorticoid treatment was initiated (mean prednisolone equivalent dose of 23.1 ± 12.7 mg/day, range 10–50). Fasting morning serum concentrations of osteocalcin (OC), amino-terminal propeptide of type I procollagen (PINP), type 1 collagen cross-linked C-telopeptide (βCTX), soluble receptor activator of nuclear factor kappaB ligand (sRANKL), osteoprotegerin (OPG), sclerostin, Dickkopf-1 (Dkk-1), and high-sensitivity C-reactive protein (hsCRP) were measured at baseline and on three consecutive days. Significant reductions in serum OC, PINP, OPG, sclerostin, and hsCRP were observed during 96 h of glucocorticoid administration, while serum βCTX showed a significant percentual increase. A significant positive correlation was found between serum concentrations of Dkk-1 and βCTX after 96 h of treatment with glucocorticoids. A significant drop in serum sclerostin, OPG, and OC observed in this study may reflect the rapid glucocorticoid-induced apoptosis of osteocytes.