Objectives: To study the interaction between cancer cells and their microenvironment such as stromal cells and extracellular matrix which influenced cancer progression. Methods: Human oral squamous carcinoma cell line, HSC-5, which derived from the primary tumor site, was cultured on plastic plate or type I collagen-coated plate in the presence or absence of conditioned media from human gingival fibroblasts. MTT, gelatin zymography and scrape wound assays were performed for the analysis of cell proliferation, gelatinase expression and cell migration, respectively. Results: The results showed that type I collagen increased cell proliferation, but decreased cell migration. Whilst the conditioned media from human gingival fibroblasts showed no effect on cell proliferation, but stimulated cell migration. Interestingly, Phenanthroline, a zinc chelator, reduced migration of HSC-5 treated with human gingival fibroblast conditioned media suggesting an involvement of MMP-dependence pathway. Moreover, type I collagen was found to decrease the expression of MMP-9, whereas conditioned media from human gingival fibroblasts increased that of both MMP-2 and MMP-9. Conclusions: These results indicated that the microenvironment played an important role in the regulation of oral squamous carcinoma cell behaviors.