• RTF exhibited excellent anti-tumor capacity both in vitro and in vivo . • RTF inhibited the invasion and migration of A549 via facilitating F-actin collapse. • RTF suppressed tumor growth via bax/bcl-2/caspase-9/caspase-3-dependent pathway. • RTF restrained VEGF expression and thereby suppressed angiogenesis in tumor. The main flavonoid components of Radix Tetrastigma (RTF) were extracted and characterized by high performance liquid chromatography. In vitro , RTF suppressed the viability of A549 cells, and inhibited the invasion and migration of A549 by damaging the lamellipodium and the structure of F-actin. In vivo , compared to the model group, RTF inhibited the growth of tumor volumes in nude mice, and decreased the weight of tumor. To uncover the inner mechanism, the proliferation-related proteins: PCNA, Ki67 and the apoptosis-related proteins: caspase-3, caspase-9, Bax and Bcl-2 were examined by immunohistochemical and western blot analysis. Compared to the model group, the production levels of PCNA, Ki67 and Bcl-2 were significantly down-regulated by RTF, and the expression levels of caspase-3, caspase-9 and Bax were markedly up-regulated. Comprehensively, RTF could suppress the proliferation of A549 and promote the apoptosis of A549, suggesting RTF as a potential resource to fight against non-small cell lung cancer (NSCLC).