Delivery of effector molecules into LMmev macrophages by enteropathogenic Escherichia coli, via its type three secretion system (T3SS), inhibits bacterial uptake by a phosphatidylinositol‐3 (PI‐3) kinase‐dependent pathway. The T3SS system, encoded by the locus of enterocyte effacement (LEE) pathogenicity island, delivers LEE‐ and non‐LEE‐encoded effector proteins into host cells. Previous studies discounted essential roles for the LEE‐encoded Map, EspF, Tir or Intimin proteins in this process but correlated it with loss of phosphorylation of the PI‐3 kinase substrate, Akt (Celli et al., 2001, EMBO J 20: 1245–1258). Given the more recent finding that these bacterial proteins are multifunctional and can act together to subvert host cellular processes, we generated a quadruple deletion mutant (Map, Tir, EspF and Intimin deficient) to unearth any cooperativity in inhibiting uptake. The quadruple mutant was as defective as the T3SS‐defective strain at preventing bacterial uptake with further studies revealing a surprising dependence on EspF but not Map, Tir or Intimin. Subversive activities previously associated with EspF are disruption of epithelial barrier function and programmed cell death, with the latter linked to EspF targeting mitochondria. Interestingly, the C‐terminal domain possesses a polyproline motif associated with protein–protein interactions. We demonstrate that EspF‐mediated inhibition of PI‐3 kinase‐dependent uptake: (i) is independent of mitochondrial targeting, (ii) requires the N‐terminal domain with and (iii) the C‐terminal domain is sufficient to disrupt barrier function but not inhibition of bacterial uptake. Moreover, loss of PI‐3 kinase‐dependent phosphorylation of Akt and gross changes in host phosphotyrosine protein profiles could not be linked to inhibition of the PI‐3 kinase‐dependent uptake process.