Guaiazulene is a plant-derived bicyclic sesquiterpene widely used as a low-toxicity anti-inflammatory agent. However, the other bioactivities and mechanisms of action of guaiazulene were not well known. Guaiazulene inhibited colony formation of the alveolar basal epithelial adenocarcinoma cell line A549 in a soft agar medium. This suggested that guaiazulene has anticancer activity. In addition, cell migration activity was evaluated using A549 and the poorly differentiated squamous cell carcinoma cell line SAS, and the results showed that guaiazulene inhibited TGF-β-induced cell migration activity. These results suggest that guaiazulene inhibits the TGF-β pathway. TGF-β induced the formation of stress fibers during cell migration, and guaiazulene inhibited stress fiber formation. Guaiazulene also inhibited phosphorylation of Focal adhesion kinase (FAK), activated during cell migration by TGF-β. These results suggest that guaiazulene inhibits cell migration by disrupting the stress fiber formation mechanism. Furthermore, TGF-β-induced changes in the expression of epithelial and mesenchymal markers were also partially inhibited by guaiazulene. These results suggest that guaiazulene may have anticancer activity and inhibit metastatic potential.