Bruceine B is a kind of quassinoid isolated from the fruits of Brucea javanica (L.) Merr. (Simaroubaceae). The anti-proliferative effects of bruceine B on NCI-H292 cells were estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the bruceine B-induced apoptosis in NCI-H292 cells was determined by fluorescence microscope through TUNEL staining with DAPI and by flow cytometry analysis via staining with Annexin V-FITC/PI. The expression levels of proteins related to apoptosis pathway were measured by western blot analysis. Our results revealed that bruceine B showed significant anti-tumor activity on lung cancer in vitro via up-regulation of p53, Bax, caspase-8 and caspase-3. Meanwhile, we also used GRP78 and caspase-12 which indicated that bruceine B induced cell death through endoplasmic reticulum (ER) stress. These results demonstrated that bruceine B induced cell apoptosis through ER stress, death receptor and mitochondria-dependent signaling pathways.