BACKGROUND: Osteosarcoma is the most common primary bone tumor in humans. It is a locally aggressive tumor at the primary site, with metastasis being the main cause of death in patients. Studies on dogs have gained prominence in oncology, as they are valuable spontaneous models of osteosarcoma. In the context of natural compounds, biotoxins are attracting increasing research interest as new therapeutic agents against cancer, such as melittin, that represents 40 to 50% of the dry weight of bee venom, and studies have already shown its antitumor effects. METHODS: We analyzed the anti-migratory and anti-invasive potential of melittin, with the wound healing and Transwell tests, apoptosis with Annexin V/IP and cell viability with the MTT test in 2D and 3D models. RESULTS: Melittin had a cytotoxic effect on osteosarcoma cell lines, with an IC50 between 1.5 and 2.5 µg/mL. In the wound healing test and Transwell test, melittin prevented cell migration and invasion, resulting in cell death due to iodide propidium marking in canine, murine and human cell lines. Melittin exhibited cytotoxicity in a 3D model of osteospheres, with a significantly higher IC50 in this type of culture, with values between 3.5 and 4.0 µg/mL. CONCLUSION: We conclude that melittin has antitumor and antimetastatic properties in canine, murine and human osteosarcoma cell lines. Consequently, we believe that further research on this promising compound will facilitate its application in the development of therapeutic agents for osteosarcoma, ultimately contributing to improved survival outcomes for cancer patients.