Abstract Pax5/BSAP is an important regulator for B lineage cell development and function. Pax5 activates many B lineage specific genes expression, suppresses lineage non-appropriate genes expression, and controls B lineage specific immunoglobulin gene VH→DJH recombination. On the other hand, abnormal Pax5 expression has been found in different type of tumors, suggesting that Pax5 might have oncogenic potential. It has been previously shown that Pax5 interferes with p53 function through inhibition p53 gene transcription. Here, we show that Pax5-inhibits p53-meidated induction of different p53-responsive reporter genes without affecting the p53 protein expression level; conversely, forced expression of p53 or activation of p53 by DNA damaging response also suppresses Pax5-mediated transcriptional activation. Co-immunoprecipitation studies provide the evidence that Pax5 interacts with p53, which is essential for the mutual functional interference between these two factors. Moreover, overexpression of Pax5 attenuates DNA damage induced p53 Ser-18 phosphorylation and inhibits p53-medaited induction of downstream target genes, including p21, Bax, Puma, and Gadd45α. Through interfering p53 function, overexpression of Pax5 protects cells from DNA damage induced apoptosis. These results reveal that Pax5 acts as a negative regulator for p53, which might be essential for the dual roles of Pax5 in B lymphopoiesis and tumorigenesis.