The cytotoxic effect of Scutebarbatine A on A549 in vitro were determined by an MTT assay and evaluated by IC50 values. Furthermore, results of Hoechst 33258 and Annexin V/PI staining assays demonstrated that Scutebarbatine A had significant antitumor effects on A549 cells via apoptosis, in a concentration-dependent manner. What's more, the mechanism was explored by western blotting, and our study revealed that Scutebarbatine A can up-regulate the expressions of cytochrome c, caspase-3 and 9, and down-regulate the levels of Bcl-2 in A549 cells. Finally, the antitumor effects of Scutebarbatine A were evaluated in vivo by using transplanted tumor nude mice, and the results confirmed that Scutebarbatine A has a notable antitumor effect on A549 cancer via mitochondria-mediated apoptosis.