Abstract Pax5/BSAP is an essential regulator for B lineage cell development and function. Pax5 activates the transcription of many B lineage specific genes, such as Cd19, Mb-1 and Blnk; controls the B lineage specific IgH VH→DJH recombination and meantime, represses the expression of other lineage inappropriate genes, such as Notch1, M-csfr, G-csfr, and Ccl3, to restrict B lineage cell development. It becomes important to understand how the Pax5 activity is regulated to exert these different functions. Here, we show that co-expression of histone acetyltransferase p300 dramatically enhances Pax5-mediated activation of a Pax5 responsive luciferase reporter construct. The p300-mediated enhancement is dependent on the intrinsic histone acetyltransferase activity of p300 and the activation domain of Pax5. Pax5 interacts with p300 in vitro and forms a complex with p300 in B lineage cells. Recombinant full length p300 purified from baculovirus or 293 cells directly acetylates purified Pax5 in vitro and overexpression of p300 induces Pax5 acetylation in 293 cells. Mass spectroscopy-based analysis identified that multiple lysine residues within the Pax5 DNA binding domain were acetylated upon coexpression of p300. Mutations of lysine 67, 87, 89, 103, and 142 residues on Pax5 attenuate p300-mediated enhancement. These results suggest that p300 acts as a potent co-activator for Pax5-mediated transcription function through acetylation of the Pax5 DNA binding domain.