Human beta-defensins (hBDs) are small, cationic antimicrobial peptides produced by oral and other mucosal epithelia. More recently, hBDs have been shown to regulate adaptive immunity. In this study, we provide new information about the potential role of hBD-3 in the progression of oral cancer. In normal human oral epithelia, hBD-3 is produced by mitotically active cells in the basal layers of oral epithelium, whereas hBD-1 and -2 are coexpressed in the differentiated spinosum and granulosum layers. Interestingly, premalignant cells in carcinoma in situ lesions overexpress hBD-3, but not hBD-1 and hBD-2, correlating with specific recruitment and infiltration of macrophages. Our in vitro studies demonstrate that hBD-3 chemoattracts THP-1 monocytic cells and that epidermal growth factor (EGF) significantly induces hBD-3 expression in oral epithelial cells via mitogen-activated protein kinase (MAPK) kinase MEK1/2, p38 MAPK, protein kinase C (PKC), and phosphoinositide 3 kinase (PI3K), but not via Janus kinase (JAK) and signal transducer and activator of transcription (STATs). These results suggest that hBD-3 serves as a mitogen responsive gene in the initiation of oral cancer and may act as a motility signal to recruit tumor-associated macrophages.