Patients taking glucocorticoid or glucocorticoid-like drugs for an extended period of time can develop osteoporosis, termed glucocorticoid-induced osteoporosis (GIOP). GIOP is the most common form of secondary osteoporosis, but the mechanism underlying its development is unclear. In the present study, we used prednisolone to treat zebrafish larvae to investigate GIOP. Our RNA deep-sequencing (RNA-seq) results show that prednisolone affects genes known to act in the extracellular region, and therefore the extracellular region, extracellular matrix, and collagen trimer might be involved in glucocorticoid-induced osteoporosis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the focal adhesion signaling pathway is the most enriched signaling pathway in terms of differentially expressed genes (DEGs). In this pathway, two adapter proteins, itga10 and itgbl1, were down-regulated in the prednisolone-treated larvae. Further experiments showed that these two genes contribute to glucocorticoid-induced osteoporosis. The results of our study provide new insights into glucocorticoid-induced osteoporosis.