Keratins 8 and 18 (K8/18) heteropolymers may regulate cell signaling via the known K18 association with 14-3-3 proteins and 14-3-3 association with Raf-1 kinase. We characterized Raf-keratin-14-3-3 associations and show that Raf associates directly with K8, independent of Raf kinase activity or Ras-Raf interaction, and that K18 is a Raf physiologic substrate. Raf activation during oxidative and toxin exposure in cultured cells and animals disrupt keratin-Raf association in a phosphorylation-dependent manner. Mutational analysis showed that 14-3-3 residues that are essential for Raf binding also regulate 14-3-3-keratin association. Similarly, Raf phosphorylation sites that are important for binding to 14-3-3 are also essential for Raf binding to K8/18. Therefore, keratins may modulate some aspects of Raf signaling under basal conditions via sequestration by K8, akin to Raf-14-3-3 binding. Keratin-bound Raf kinase is released upon Raf hyperphosphorylation and activation during oxidative and other stresses.