Laminin-5 (Ln-5) is expressed in several human carcinomas and hypothesized to contribute to tumor invasion. To understand the role of Ln-5 in human cancers, we stably delivered small interfering RNAs (siRNAs) directed against the Ln-5 γ2 chain into JHU-022-SCC cells (022), a non-invasive oral squamous cell carcinoma (OSCC) cell line which secretes Ln-5. Lysates from γ2 siRNA cells (022-siγ2) had nearly undetectable levels of the γ2 chain while the α3 and β3 subunits of Ln-5 remained unchanged compared to parental and control. In conditioned medium from 022-siγ2 cells, the γ2 chain and the Ln-5 heterotrimer were barely detectable, similar to an invasive OSCC cell line. Conditioned medium from 022-siγ2 cells contained less α3 and β3 subunits than both parental and control. Although the proliferation and adhesive properties of the 022-siγ2 cells remained similar to parental and control cells, 022-siγ2 cells showed increased detachment and a fibroblastic morphology similar to invasive cells. Moreover, migration, in vitro invasion, and in vivo tumorigenicity were enhanced in 022-siγ2 cells. Our results suggest that the Ln-5 γ2 chain regulates the secretion of the α3 and β3 subunits. More importantly, suppression of Ln-5 results in a phenotype that is representative of invasive tumor cells.