Enteropathogenic Escherichia coli (EPEC) virulence is correlated with intimate adherence to gut epithelial cells, loss of absorptive microvilli and reorganization of host cytoskeletal proteins into pedestal-like structures beneath the adherent bacteria. These processes depend on Tir (i) being inserted into the plasma membrane; (ii) being tyrosine phosphorylated; and (iii) interacting with the bacterial outer membrane protein, intimin. However, phosphorylation on other undefined residues leads to approximately 5 kDa and approximately 2 kDa increases in Tir apparent molecular mass within host cells. In this study, we show that equivalent shifts can be induced in vitro by phosphorylation of Tir on two serine (S434 and S463) residues by protein kinase A (PKA). Our data suggest that the sequential addition of two phosphate groups triggers conformational changes in Tir structure that may supply the energy to insert Tir into the plasma membrane. PKA was also shown to modify Tir within host cells on S434 to induce the approximately 5 kDa shift. Whereas modification of S434 was not essential to generate an actin-nucleating molecule, it was required for Tir to induce pedestal elongation efficiently. This study not only increases our understanding of the mechanism by which phosphorylation induces shifts in Tir apparent molecular mass and suggests a mechanism by which Tir may be inserted into the plasma membrane, but also reveals a role for non-tyrosine phosphorylation in Tir function and identifies the first kinase that can modify Tir in vitro or in vivo.