7,7"-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from Taxus x media cv. Hicksii, induces apoptotic and autophagic cell death. However, whether DMGF suppresses tumor metastasis is unclear. The aim of this study was to investigate the anti-metastatic activities of DMGF on the metastatic processes of melanoma cells in vivo and in vitro. A transwell assay showed that DMGF could effectively attenuate the motility of B16F10 cells, and the results of real-time PCR revealed that DMGF also suppressed the expressions of matrix metalloproteinase-2 (MMP-2). Moreover, DMGF did not influence tube formation but inhibited the migration of endothelial cells. Furthermore, animal models were used to monitor the effects of DMGF on tumor metastasis, and all models showed that DMGF significantly suppressed the metastatic behaviors of B16F10 cells, including intravasation, colonization, and invasion of the lymphatic duct. In addition, DMGF could also reduce the densities of the blood vessels in the tumor area in vivo. Further investigation of the molecular mechanisms of anti-metastatic activity revealed that DMGF can down-regulate the levels of key modulators of the Cdc42/Rac1 pathway to interfere in F-actin polymerization and suppress the formation of lamellipodia by reducing the phosphorylation of CREB. These data suggested that DMGF presents anti-metastatic activities in B16F10 melanoma cells. Here, we demonstrated that DMGF can inhibit the metastasis of highly invasive melanoma cancer cells through the down-regulation of F-actin polymerization. Considering these findings, DMGF may be further developed to serve as a chemoprevention drug for patients with metastatic melanoma.