The objective of this study was to investigate the impact of human beta-defensins (hBDs) on oral squamous cell carcinoma (OSCC) proliferation and hBD expression in vitro. BHY-OSCC cell lines were stimulated with hBD-1, -2, and -3. Proliferation of BHY cells was ascertained and hBD-mRNA expression was evaluated by real-time PCR. Proliferation of BHY cells decreased by 25% in response to hBD-1 stimulation but increased after stimulation with hBD-2 and -3. HBD-1 stimulation enhanced hBD-3 expression, whereas HBD-2 stimulation decreased early hBD-3 expression. HBD-3 stimulation enhanced hBD-1 expression. HBDs profoundly impact on OSCC proliferation and hBD expression in vitro. Therefore, hBD-1 might function as a tumor suppressor gene in OSCCs, while hBD-2 and -3 might be protooncogenes.