Osteonecrosis of the femoral head (ONFH) is a devastating musculoskeletal disease characterized by the impaired circulation of bone. The purpose of this study was to explore the underlying mechanisms of the protective effect of icariin on the glucocorticoid-induced injury of bone microvascular endothelial cells (BMECs). Normal BMECs were extracted from the femoral heads by enzymatic isolation and magnetic-activated cell sorting methods. Dexamethasone and icariin were used to intervene BMECs in microfluidic organ chips, and phalloidin staining was conducted to observe the cell morphology and viability. Then next-generation transcriptome sequencing and real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were performed to identify the differentially expressed genes (DEGs) in different groups. Through the microfluidic organ chip, it can be observed that after dexamethasone intervention, the filamentous structure in cell fibers disappeared and the cell morphology changed from spindle to round until death. Icariin could relieve these changes and showed a protective effect on glucocorticoid-damaged BMECs. In addition, 201 DEGs were detected between the icariin protection group and the dexamethasone group, which were significantly enriched in 17 signaling pathways. 8 of the top ten selected hub genes (IL6, PTGS2, VEGFA, etc.) were confirmed by qRT-PCR. Transcription factors (TFs)-gene network showed 63 connections between 18 TFs and 12 DEGs. For instance, GATA2 could regulate 5 DEGs. The associations between 92 miRNA and 12 DEGs were visualized in a miRNA-gene network. The hub miRNA, has-mir-335-5p was predicted to interact with 8 DEGs (PTGS2, VEGFA, etc.). Microfluidic organ chips could provide excellent morphological results for cell experiments, by which it could be observed that icariin showed a protective effect on the glucocorticoid-induced injury of BMECs. Beside, these DEGs, possible regulatory TF (GATA2, FOXC1, etc.) and miRNA (has-mir-335-5p) might be dysregulated in the initiation of ONFH and have prospective importance in ONFH diagnosis and therapy. (C) 2022 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.