EBP50 is a highly phosphorylated scaffold protein which participates in various cellular signalling pathways. Several protein kinases phosphorylate EBP50 at specific serine residues and the phosphorylation status of EBP50 is greatly related to its functional regulation. By site-directed mutagenesis and a phospho-specific antibody, we confirmed a protein kinase C (PKC)-dependent phosphorylation of EBP50 at canine serine 347 and 348. Increased phosphorylation at these two serine residues led to the relocation of EBP50 from plasma membrane to cytosol, and the dissociation of EBP50 from ezrin and β-PIX, which two upstream regulator of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to Rac1 activation. Further, detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation at serine 347 and 348, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation are remarkably resistant to apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function.