Berberine (BBR), an isoquinoline derivative alkaloid compound, has been reported to have anti-oxidant and anti-carcinogenic effects. A loss of functional p53 is involved with an increased risk of cancer proliferation and metastasis. Here, we investigated the effect of BBR on the transcriptional activity and the protein expression of p53 in p53-positive (wild- type, MCF7 cells) and p53-negative (mutant, MDA-MB231 cells) human breast cancer cells. Our results showed that the basal level of p53 mRNA and protein expression was increased by BBR treatment. However, tumor promoter, TPA, decreased the level of p53 mRNA and protein expression in MCF7 cells with wild-type p53. In addition, TPA-induced down-regulation of p53 mRNA and protein expression was increased by UO126, but not by SP600125 and SB203580. To verify the regulatory mechanism of p53 protein expression, we investigated the effects of proteasomal inhibitors (ALLN and MG132) or a lysosomal inhibitor (chloroquine) on TPA-induced down-regulation of p53. We observed that TPA-induced down-regulation of p53 protein was prevented by ALLN and MG132, but not by chloroquine. Further, we investigated the effect of BBR on TPA-induced down-regulation of p53 mRNA and protein levels. Interestingly, the levels of TPA-induced down-regulation of p53 mRNA and protein were prevented by BBR, but MDA-MB231 cells with mutated p53 were not affected. In addition, TPA-induced down-regulation of p53 mRNA was also prevented by BBR. Taken together, we suggest that BBR may be used as an effective ingredient for anticancer products, which trigger the transcriptional activity and the inhibition of the degradation of p53, a tumor suppressor gene, in human breast cancer.