Calycosin is reportedly evidenced with pharmacologically treating bone cells. However, the comprehensive anti-osteosarcoma (OS) mechanisms of calycosin have not been uncovered. By using a systemic method of network pharmacology, the present study aimed to reveal potential anti-OS biotargets and molecular mechanisms played by calycosin. Moreover, human and animal experiments were conducted to verify the core biotargets of calycosin against OS. As results, all primary and core biotargets, biological processes, molecular pathways of calycosin against OS were revealed. Additionally, top 20 biological processes and pathways of calycosin against OS were identified. In human study, the OS sections resulted in reduced expressions of tumor protein p53 (TP53), Caspase-3 (CASP3), and elevated X-linked inhibitor of apoptosis protein (XIAP) expression in comparison with OS-free controls. As shown in cell culture study, calycosin-treated OS cells showed reduced cell proliferation, and promoted cell apoptosis. In TUNEL stains, calycosin resulted in elevated apoptotic cells. As showed in immunostaining, calycosin-treated OS cells exhibited intracellular up-regulation of TP53, CASP3 expressions, and decreased XIAP expressions. Taken together, the biological informational findings manifest the candidate and core biotargets, molecular functions and pathways of calycosin against OS. Attractively, these core biotargets may be used for effectively detecting and treating human OS.