Tetramethylpyrazine (TMPZ) is one of the active compounds extracted from the traditional Chinese herb Chuanxiong. Several studies have shown its anti-cancer properties. However, its functions in lung cancer and the underlying cellular mechanisms are relatively unknown. Our present study aimed to investigate the effects of TMPZ on A549 and 95D cells. The MTT assay showed that TMPZ decreased cell viability in a dose- and time-dependent manner. The results of the colony formation assay indicated that TMPZ strongly suppressed colony formation ability in A549 and 95D cells. Flow cytometric analysis revealed that TMPZ induced S phase arrest in lung cancer cells. In addition, TMPZ induced apoptosis, as shown by the results of propidium iodide/Annexin V double-staining. Furthermore, TMPZ decreased mitochondrial membrane potential (∆Ψm) in a dose-dependent manner. Finally, western blot analysis of TMPZ-treated cells revealed the activation of Caspase-3 and the increase of the ratio of Bax/Bcl-2. These results demonstrated that TMPZ could suppress carcinogenesis of lung cancer cells through blocking cell cycle and inducing mitochondria-dependent apoptosis by regulating Caspase-3 and Bax/Bcl-2, suggesting that TMPZ may be a promising drug to treat lung cancer.