Deguelin, a rotenoid of the flavonoid family, has been reported to possess antiproliferative and anticarcinogenic activities in several cell lines and tumor models. However, it is still unclear whether deguelin effectively inhibits tumor-associated lymphangiogenesis and lymphatic metastasis. Since tumor production of vascular endothelial cell growth factor (VEGF)-D was associated with tumor lymphangiogenesis and lymphatic metastasis, we established the mouse lymphatic metastasis model by transfecting high expression VEGF-D into LL/2 Lewis lung cells (VEGF-D-LL/2) and explored the effects of deguelin on lymphatic metastasis in the immunocompetent C57BL/6 mice. Our results indicated that deguelin inhibited proliferation, migration of VEGF-D-LL/2 cells via downregulating AKT and mitogen-activated protein kinase pathway and interfered tube formation of lymphatic vascular endothelial cells on matrigel at nanomolar concentrations. Deguelin significantly downregulated the expression of VEGF-D both at mRNA and protein levels in VEGF-D-LL/2 cells in a dose-dependent manner. In the in vivo study, intraperitoneal administration of deguelin (4 mg/kg) remarkably inhibited the tumor-associated lymphangiogenesis and lymphatic metastasis. The rates of lymph node and lung metastasis in deguelin-treated mice were 0 and 16.7% compared with 58.3 and 83.3% in control group mice, respectively. Deguelin also resulted in a remarkable delay of tumor growth and prolongation of life span. Immunohistochemical staining with antibodies against VEGF-D, LYVE-1 and VEGFR-3 revealed fewer positive vessel-like structures in deguelin-treated mice compared with control group mice. Taken together, we demonstrate for the first time that deguelin suppresses tumor-associated lymphangiogenesis and lymphatic metastasis by downregulation of VEGF-D both in vitro and in vivo.