To clarify the roles of integrin and extracellular matrix (ECM) in the process of non-small cell lung cancer (NSCLC) brain metastasis, we established an in vivo model of brain metastasis of human NSCLC cell line EBC-1/original in athymic mice, and established highly brain metastatic subclone EBC-1/brain and highly bone metastatic subclone EBC-1/bone. Integrin expression of these subclones was evaluated by flow cytometry. In vitro cell attachment, migration and proliferation assays with ECMs were performed using these subclones. Expression of integrin alpha3 subunit was higher in EBC-1/brain than in both EBC-1/original and EBC-1/bone. In vitro cell attachment, migration, and proliferation assays revealed that EBC-1/brain had higher affinity and higher reactivity to laminin than EBC-1/original and EBC-1/bone. Blocking of integrin alpha3beta1 significantly (P < 0.05) decreased brain metastasis by EBC-1/brain. Interaction of integrin alpha3beta1 and laminin plays important roles in the process of brain metastasis of non-small cell lung cancer.