Death-domain-associated protein (DAXX) is a multifunctional protein that regulates a wide range of cellular signaling pathways for both cell survival and apoptosis. Regulation of DAXX gene expression remains largely obscure. We recently reported that berberine (BBR), a natural product derived from a plant used in Chinese herbal medicine, downregulates DAXX expression at the transcriptional level. Here, we further investigate the mechanisms underlying the transcriptional suppression of DAXX by BBR. By analyzing and mapping the putative DAXX gene promoter, we identified the core promoter region (from -161 to -1), which contains consensus sequences for the transcriptional factors Sp1 and Ets1. We confirmed that Sp1 and Ets1 bound to the core promoter region of DAXX and stimulated DAXX transcriptional activity. In contrast, BBR bound to the DAXX core promoter region and suppressed its transcriptional activity. Following studies demonstrated a possible mechanism that BBR inhibited the DAXX promoter activity through blocking or disrupting the association of Sp1 or Ets1 and their consensus sequences in the promoter. Downregulation of DAXX by BBR resulted in inhibition of MDM2 and subsequently, activation of p53, leading to cancer cell death. Our results reveal a novel possible mechanism: by competitively binding to the Sp1 and Ets1 consensus sequences, BBR inhibits the transcription of DAXX, thus inducing cancer cell apoptosis through a p53-dependent pathway. Laboratory Investigation (2013) 93, 354-364; doi:10.1038/labinvest.2012.172; published online 7 January 2013