Upon transition from the exponential (E) to the post-exponential phase (PE) of growth, Legionella pneumophila undergoes a phenotypic modulation from a replicative to a highly infectious form. This transition requires a delicate regulatory cascade that is triggered to induce expression of various virulence-related genes. We have recently characterized eleven L. pneumophila eukaryotic-like ankyrin effectors (Ank) shared between the four sequenced genomes of L. pneumophila. The AnkB effector recruits polyubiquitinated proteins to the Legionella-containing vacuole (LCV). It is not known whether expression of the ank genes is regulated by various regulators triggered at the PE phase and whether this regulation is essential for function. Here we show that temporal and differential regulation of the ank genes is mediated by RelA, the enhancer protein LetE, and the two component systems LetA/S and PmrA/B. Consistent with the expression of ankB at the PE phase, we show that bacteria grown to the PE but not the E phase recruit polyubiquitinated proteins to the LCV within Acanthamoeba in an AnkB-dependant mechanism. We conclude that the genes encoding the eukaryotic-like Ank effectors of L. pneumophila are temporally and spatially regulated at the PE phase.