ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus membranaceus, commonly known as Huangqi in China, is a traditional herbal medicine that has attracted significant attention for its immunomodulatory effects. It has been widely studied in various clinical contexts, including cancer treatment. Astragalus injection (HQI) is clinically used for treating myocarditis and cardiac insufficiency. However, its potential therapeutic effects on osteosarcoma, a highly aggressive bone tumor, remain largely unexplored. AIM OF THE STUDY: The aim of this study was to investigate the potential therapeutic effects of HQI on osteosarcoma and to elucidate its underlying mechanisms of action. Specifically, we aimed to determine whether HQI could inhibit osteosarcoma growth in vivo, identify its key active components and molecular targets, and explore its immunomodulatory effects on the tumor microenvironment. MATERIALS AND METHODS: Mice with osteosarcoma were treated with HQI, and tumor growth was monitored. The number of CD8 RESULTS: Our study found that HQI significantly suppresses osteosarcoma growth in vivo by increasing the number of CD8 CONCLUSION: Our study demonstrated that HQI, particularly its active component CG, holds potential as a therapeutic agent for osteosarcoma. The primary mechanism underlying its anti-osteosarcoma effects involves modulating the immune response and targeting CTSL. These findings provide a scientific basis for the development of HQI as a novel immunomodulatory therapy for osteosarcoma.