The correlation between cigarette smoking and the onset of non-small cell lung cancer is well established. Direct effects of exposure to nicotine (the active component in cigarettes) include increased proliferation, angiogenesis induction, and resistance to apoptosis. Parthenolide (PTH) is a sesquiterpene lactone with anticancer properties against several cancer types. In this work, we tested the ability of PTH to inhibit the proliferating effect of nicotine in lung cancer cell lines. MTT assay was used to measure cell survival of A549 and H526 cells exposed to nicotine, PTH, and their combination. VEGF detection kit was used to measure angiogenesis inhibition while apoptosis induction was evaluated by measuring caspase-3 activity. Real time PCR assay was used to detect the change in expression of several genes associated with cell proliferation and apoptosis. PTH inhibited lung cancer cells in a concentration-dependent manner and decreased the proliferation stimulating effect of nicotine. Caspase-3 activity and VEGF assays evidenced an apoptosis-inducing and VEGF- inhibiting effects of PTH. The real time PCR assay demonstrated that PTH down-regulated the expression of Bcl-2 and up-regulated the expression of E2F1, P53, GADD45, BAX, BIM, and CASP 3,7,8,9, which indicates an activation of P53- dependent apoptosis pathway. Furthermore, this pathway remained active in the presence of nicotine suggesting the ability of PTH to exclude the anti-apoptotic effect of nicotine. Our results indicate that PTH inhibits nicotine proliferating effect on lung cancer. The anticancer effect of PTH is mediated by angiogenesis inhibition and activation of P53- dependent apoptosis. PTH is a promising natural product for inhibiting and treating nicotine-associated lung cancer. However, further studies on more lung cancer cell lines and on protein level are needed to fully understand its mechanisms of action.