Protease-activated receptor 1 (PAR1) and integrins play an important role in thrombin-mediated tumor cell invasion. However, the role of PAR1 and integrin ανβ5 and the relationship between the two receptors in thrombin-induced lung cancer invasion remains unknown. Moreover, the mechanisms through which immobilized thrombin facilitates tumor invasion are poorly understood. In this study, both native and immobilized thrombin promoted lung cancer cell adhesion, migration and extracellular signal-regulated kinase phosphorylation. Integrin ανβ5 is involved in both native and immobilized thrombin-mediated tumor cell invasion; PAR1 had no effect on immobilized thrombin-mediated cell invasion. PAR1 and integrin ανβ5 colocalized on the surface of native thrombin-treated cells. This study suggests that targeting of integrin ανβ5 or the PAR1-integrin ανβ5 complex may present an important therapeutic opportunity to prevent lung cancer invasion.