Heat Shock Factor 1 (HSF1) monitors the structural integrity of the proteome. Phosphorylation at S326 is a hallmark for HSF1 activation, but the identity of the kinase(s) phosphorylating this site has remained elusive. We show that the dietary agent phenethyl isothiocyanate (PEITC) inhibits heat shock protein 90 (Hsp90), the main negative regulator of HSF1, activates p38 MAPK, increases S326 phosphorylation, trimerization and nuclear translocation of HSF1, and the transcription of a luciferase reporter as well as the endogenous prototypic HSF1 target Hsp70. In vitro, all members of the p38 mitogen-activated protein kinase (MAPK) family rapidly and stoichiometrically catalyze the S326 phosphorylation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPK, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation, and unexpectedly, revealed that p38 MAPK also catalyze phosphorylation of HSF1 at S303/307, previously known repressive post-translational modifications. Thus, we have identified p38 MAPK as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPK are important determinants of the extent and duration of the heat shock response.