Background and Purpose: Osteosarcoma presents significant clinical challenges due to its aggressive nature and propensity for metastasis, prompting the investigation of alternative treatment strategies. CBD has demonstrated potential in cancer treatment. This study aims to elucidate the effect of CBD on the malignant phenotype of osteosarcoma and to investigate its molecular mechanisms. Experimental Approach: The CCK-8 assay was employed to identify the effective concentration of CBD, and its impact on the malignant phenotype of osteosarcoma cells was investigated using EdU proliferation assays, Transwell experiments, and scratch assays. A mouse xenograft tumor model was established for in vivo observation. Utilizing network pharmacology and RNA-seq data, key pathways and targets were predicted and subsequently validated through ELISA, RT-qPCR, and Western blot techniques. To ascertain the targets of CBD action, molecular docking, CETSA, and SPR experiments were subsequently conducted. Key Results: CBD significantly inhibits the activity and proliferation, migration, and invasion capabilities of osteosarcoma cells, and suppresses the growth of transplanted tumors in nude mice in vivo. CBD is involved in the regulation of inflammation and the NF-κB signaling pathway in osteosarcoma cells. p65 and CCL5 form a positive feedback loop, and CBD directly targets p65, effectively disrupting the transcriptional activation of the NF-κB signaling pathway mediated by TNF-α. Conclusion and Implications: Our findings for the first time indicate that CBD inhibits the malignant progression of osteosarcoma by targeting and regulating the TNF-α/NF-κB/CCL5 chemokine axis, disrupting its coordinated inflammatory cascade. This highlights CBD’s potential as a novel therapeutic agent for osteosarcoma.