Background: With the aging of the world's population, the incidence of osteoporosis (OP) has become a public health problem of worldwide concern. Research shows that icariin may have a therapeutic effect on OP. Material/Methods: PharmMapper was utilized to predict the potential targets of icariin. GeneCards and Online Mendelian Inheritance in Man (OMIM) were used for the collection of OP genes. The STRING database was utilized to obtain the protein-protein interaction (PPI) data. We used Cytoscape 3.7.2 to construct and analyze the networks. The genes and targets in the networks were input into the Database for Annotation, Visualization and Integrated Discovery (DAVID) to undergo Gene Ontology (GO) and pathway enrichment analysis. Finally, animal experiments were performed to verify the prediction results of this study. Results: A total of 297 icariin potential targets and 262 OP genes were obtained, and an icariin-OP PPI network was constructed and analyzed. The results of the GO enrichment analysis showed that icariin can regulate the steroid hormone-mediated signaling pathway, skeletal system development, extracellular space, cytosol, and steroid hormone receptor activity. The results of the pathway enrichment analysis showed that icariin can regulate osteoclast differentiation, FoxO, estrogen, and PPAR signaling pathways. The results of the experiments showed that icariin can increase estradiol, b-catenin, and Receptor Activator of Nuclear Factor-k B Ligand (RANKL)/osteoprotegerin (OPG) ratio in postmenopausal OP rats (P<0.05). Conclusions: This research found that the icariin can regulate OP-related biological processes, cell components, molecular functions, and signaling pathways. © Med Sci Monit