Human β-defensin 1 (hBD-1) is an important antibacterial polypeptide whose expression is not induced by infection or inflammation. Our objective was to study the regulation of hBD-1 expression. Recently, we found that albumin up-regulated hBD-1 as well as c-Myc expression, suggesting that c-Myc may regulate hBD-1 expression via a non-inflammatory pathway. Direct evidence for the involvement of c-Myc was achieved by the inhibition of hBD-1 expression in the presence of a specific c-Myc inhibitor. Since both c-Myc and CLOCK:BMAL1 heterodimer, the complex of the core clock mechanism, bind to E-box (5′-CACGTG-3′) and E-box-like sequences to activate transcription, we studied whether hBD-1 expression was also regulated by the biological clock. Synchronization of HCT-116 cells by dexamethasone showed oscillation of hBD-1 and c-myc mRNA indicating that both are clock-controlled output genes. Using transfections and luciferase reporter assays in human embryonic kidney (HEK-293) cells, we found that hBD-1 promoter was induced by CLOCK:BMAL1 co-expression. hBD-1 promoter truncation and mutagenesis analyses revealed that the distal E-box-like binding sequence was the target of both CLOCK:BMAL1 and c-Myc for hBD-1 expression. This activation was abolished when CRY1 was co-expressed in these cells. Thus, hBD-1 expression is mediated by c-Myc and the CLOCK:BMAL1 heterodimer, whereas CRY1 expression represses this complex. These changes in hBD-1 levels lead to its circadian oscillation.