Matrine is an alkaloid isolated from Sophora flavescens and shows anticancer activities. The present study was carried out to determine the cytotoxic effects of matrine on cisplatin-resistant non-small cell lung cancer (NSCLC) cells and the associated molecular mechanisms. Parental and cisplatin-resistant A549 and H460 NSCLC cells were treated with 1 or 2 g/l of matrine for 48 h, and cell viability and apoptosis were assessed. β-catenin-mediated transcriptional activity, mitochondrial membrane potential (ΔΨm) changes, activation of caspases, and survivin expression were examined. The effect of overexpression of survivin on the anticancer activity of matrine was investigated. Compared to the parental cells, cisplatin-resistant NSCLC cells showed increased β-catenin transcriptional activity. Matrine treatment resulted in a significant reduction in β-catenin activation and survivin expression in the cisplatin-resistant cells. Matrine caused apoptotic death in the cisplatin-resistant NSCLC cells, coupled with loss of ΔΨm and activation of caspase-9 and-3. Matrine-induced apoptosis of the cisplatin-resistant NSCLC cells was significantly reversed by overexpression of survivin. In conclusion, matrine exposure induces mitochondrial apoptosis in cisplatin-resistant NSCLC cells, which is largely mediated through inactivation of β-catenin/survivin signaling. Further investigation of the therapeutic benefit of matrine in overcoming cisplatin resistance in NSCLC is warranted.