Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), which belongs to the EGF family, has been shown to stimulate the growth of a variety of cells in an autocrine or paracrine manner. Although HB-EGF is widely expressed in tumors when compared to normal tissue, its contribution to tumor invasion is still not known. In the present study, we analyzed the effects of HB-EGF on the invasion activity of a cultured oral cancer cell line using short interfering RNA (siRNA).Oral squamous carcinoma cell lines, HSC3 and SAS, were transfected with siRNA targeting HB-EGF. Expression of HB-EGF was analyzed by real-time PCR. The invasiveness of the transfected cells was determined using a matrigel invasion assay, and MMP-9 production was measured by RT-PCR and gelatin zymography.The expression of HB-EGF was reduced in HSC3-siRNA and SAS-siRNA cells. The matrigel invasion assay demonstrated that the invasiveness of HSC3-siRNA and SAS-siRNA cells was reduced. Gelatin zymography demonstrated that in HSC3-siRNA and SAS-siRNA cells, MMP9 production was decreased.These findings suggest that HB-EGF expression is related to the invasion activity of oral cancer, particularly via regulation of MMP9.