Tip60 is a multifunctional acetyltransferase involved in multiple cellular functions. Acetylation of p53 at K120 by Tip60 promotes p53-mediated apoptosis after DNA damage. We previous showed that Tip60 activity is induced by phosphorylation at T158 by p38. In this study, we investigated the role of p38-mediated Tip60 phosphorylation in p53-mediated, DNA damage-induced apoptosis. We found that DNA damage induces p38 activation, Tip60-T158 phosphorylation, and p53-K120 acetylation with similar kinetics. p38α is essential for DNA damage-induced Tip60-T158 phosphorylation. In addition, both p38α and Tip60 are essential for p53-K120 acetylation, binding of p53 to PUMA promoter, PUMA expression and apoptosis induced by DNA damage. Moreover, DNA damage induces protein kinase activity of p38α towards Tip60-T158, and constitutive activation of p38 in cells leads to increases in Tip60-T158 phosphorylation, p53-K120 acetylation, PUMA expression and apoptosis. Furthermore, the Tip60-T158A mutant that cannot be phosphorylated by p38 fails to mediate p53-K120 acetylation, PUMA induction, and apoptosis following DNA damage. These results establish that Tip60-T158 phosphorylation by p38 plays an essential role in stimulating Tip60 activity required for inducing the p53-PUMA pathway that ultimately leads to apoptosis in response to DNA damage, which provides a mechanistic basis for the tumor-suppressing function of p38 and Tip60.