The matrix glycoprotein, fibronectin, stimulates the proliferation of non-small cell lung carcinoma in vitro through α5β1 integrin receptor-mediated signals. However, the true role of fibronectin and its receptor in lung carcinogenesis in vivo remains unclear. To test this, we generated mouse Lewis lung carcinoma cells stably transfected with short hairpin RNA shRNA targeting the α5 integrin subunit. These cells were characterized and tested in proliferation, cell adhesion, migration, and soft agar colony formation assays in vitro. In addition, their growth and metastatic potential was tested in vivo in a murine model of lung cancer. We found that transfected Lewis lung carcinoma cells showed decreased expression of the α5 gene, which was associated with decreased adhesion to fibronectin and reduced cell migration, proliferation, and colony formation when compared with control cells and cells stably transfected with α2 integrin subunit in vitro. C57BL/6 mice injected with α5-silenced cells showed lower burden of implanted tumors, and a dramatic decrease in lung metastases resulting in higher survival as compared with mice injected with wild-type or α2 integrin-silenced cells. These observations reveal that recognition of host- and/or tumor-derived fibronectin via α5β1 is important for tumor growth both in vitro and in vivo, and unveil α5β1 as a potential target for the development of anti-lung cancer therapies.