The roots of Trichosanthes kirilowii (TK) have been used in traditional oriental medicine for the treatment of respiratory diseases. In this study, we investigated whether an ethanolic root extract of TK (ETK) can regulate the metastatic potency of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant human lung cancer cells. The relative migration and invasion abilities of erlotinib-resistant PC9 (PC9/ER) and gefitinib-resistant PC9 (PC9/GR) cells were higher than those of parental PC9 cells. Mesenchymal markers were overexpressed, whereas epithelial markers were downregulated in resistant cells, suggesting that resistant cells acquired the EMT phenotype. ETK reduced migration and invasion of resistant cells. The expression levels of N-cadherin and Twist were downregulated, whereas Claudin-1 was upregulated by ETK, demonstrating that ETK suppresses EMT. As a molecular mechanism, Src was dephosphorylated by ETK. The anti-metastatic effect of ETK was reduced by transfecting PC9/ER cells with a constitutively active form of c-Src. Dasatinib downregulated N-cadherin, Twist, and vimentin, suggesting that Src regulates EMT in resistant cells. Notably, CuB played a key role in mediating the anti-metastatic activity of ETK. Collectively, our results demonstrate that ETK can attenuate the metastatic ability of EGFR-TKI-resistant lung cancer cells by inhibiting Src-mediated EMT.