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AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (Δloop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidDΔloop phenocopied a L. pneumophila ΔsidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection. Author summary: The frequency of Legionnaires' disease outbreaks, a serious pneumonia caused by the intracellular pathogen Legionella pneumophila, has increased more than 4-fold between the years 2000 and 2015 [CDC, Summary of Notifiable Diseases, United States, 2015]. L. pneumophila secretes hundreds of bacterial effectors to manipulate host cell processes. Rab GTPases, which control intracellular vesicle trafficking in eukaryotes, are a known target of Legionella effectors. SidM and SidD both target host cell Rab1, but with opposing activities. While SidM catalyzes the attachment of adenosine monophosphate (AMP) to Rab1, SidD catalyzes AMP removal from Rab1, a process known as deAMPylation. Here we present the crystal structure of full length SidD, including the previously uncharacterized C-terminal domain (CTD). We discovered that the CTD assumed a unique fold that was critical for membrane localization of SidD within host cells, and that this localization was dependent on two structural elements: a stretch of hydrophobic amino acid residues that represents a general membrane targeting determinant, and a two-helix bundle that functions as the specificity determinant. Deletion of either of these elements interfered with the correct localization of SidD within mammalian cells, confirming that positioning of bacterial effectors is critically important for their biological function. [ABSTRACT FROM AUTHOR]