Considering the high metastatic potential of osteosarcoma, not only pro-apoptosis, but also anti-metastasis is important for anti-osteosarcoma therapy. Previously, the authors reported the pro-apoptotic and tumor-inhibitory effects of theabrownin (TB) on osteosarcoma cells; however, its effects on the metastasis-related migration and invasion of osteosarcoma cells remain unknown. The present study conducted RNA sequencing (RNA-seq) on xenograft zebrafish samples and performed in vitro experiments, including RT-qPCR, cell viability analysis, clone formation assay, cell cycle analysis, immunofluorescence, cell migration assay, cell invasion assay, wound healing assay and western blot (WB) analysis to evaluate the anti-metastatic effects and mechanism of TB against osteosarcoma cells. The RNA-seq data revealed that TB significantly downregulated the expression of genes involved in the microtubule bundle formation of U2OS cells, which was verified by RT-qPCR. The cell viability and clone formation data indicated that TB significantly inhibited U2OS cell viability and colony numbers. The results of cell cycle analysis revealed the blocked cell cycle progression of U2OS by TB. The immunofluorescent data revealed an evident cytoskeleton-inhibitory effect of TB against the microfilament and microtubule formation of U2OS cells. The results of cell migration and invasion demonstrated that TB significantly inhibited U2OS cell migration and invasion. The results of WB analysis revealed that TB significantly regulated key molecules of epithelial-mesenchymal transition [EMT; e.g., E-cadherin, vimentin, Snail-1, Slug and zinc finger E-box-binding homeobox 1 (ZEB-1)] and those of the nuclear factor (NF)-κB pathway (e.g., NF-κB, phospho-IKKα and phospho-IKKβ), indicating that NF-κB pathway-related EMT suppression may mediate the mechanisms underlying the anti-migratory and anti-invasive effects of TB against osteosarcoma. To the best of our knowledge, this is the first study on the inhibitory effects and mechanisms of TB on the cytoskeleton-dependent cell cycle, migration and invasion of human osteosarcoma cells. The findings presented herein suggest that TB may be a promising anti-metastatic candidate for anti-osteosarcoma therapy.