Lung cancer is one of malignant tumors that cause great threats to human health, which causes the fastest growing morbidity and mortality. Oleuropein as natural production exerts anticancer effects in several cancer cells. In the study, we investigated apoptotic effect of oleuropein on A549 cells and the underlying mechanisms. Oleuropein markedly decreased cell viability in A549 cells by resulting in G₂/M phase arrest, but failed to decreased cell viability in BEAS–2B cells significantly. Apoptosis by oleuropein was confirmed by apoptotic morphology, accumulation in a sub-G₁ peak, nucleus fragmentation and cleavage of PARP. Dose-dependent elevation in p-p38MAPK and p-ATF-2 was observed whereas apparent changes could not be observed in p-JNK and p-c-Jun, showing activation of p38MAPK but not JNK. Interestingly, ERK1/2 appeared to be constant while p-ERK1/2 was reduced dose-dependently. Oleuropein caused decrease in mitochondrial membrane potential, increase in Bax/Bcl- 2 ratio, release of mitochondrial cytochrome c and activation of caspase-9 and caspase-3, implying that mitochondrial apoptotic pathway was activated. Additionally, oleuropein-induced apoptosis was dramatically attenuated by Z-VAD-FMK (caspase inhibitor). The p38MAPK inhibitor prevented production of apoptotic bodies and reduced expressions of cleaved-PARP, p-P38, p-ATF-2 and release of cytochrome c. Taken together, these results demonstrated p38MAPK signaling pathway mediated oleuropein-induced apoptosis via mitochondrial apoptotic cascade in A549 cells. Oleuropein has the potential to be a therapeutic drug for lung cancer treatment.