The amoeba-resistant bacteriumLegionella pneumophilacauses Legionnaires’ disease and employs a type IV secretion system (T4SS) to replicate in the unique, ER-associatedLegionella-containing vacuole (LCV). The large fusion GTPase Sey1/atlastin is implicated in ER dynamics, ER-derived lipid droplet (LD) formation, and LCV maturation. Here, we employ cryo-electron tomography, confocal microscopy, proteomics, and isotopologue profiling to analyze LCV-LD interactions in the genetically tractable amoebaDictyostelium discoideum. Dually fluorescence-labeledD. discoideumproducing LCV and LD markers revealed that Sey1 as well as theL. pneumophilaT4SS and the Ran GTPase activator LegG1 promote LCV-LD interactions. In vitro reconstitution using purified LCVs and LDs from parental or Δsey1mutantD. discoideumindicated that Sey1 and GTP promote this process. Sey1 and theL. pneumophilafatty acid transporter FadL were implicated in palmitate catabolism and palmitate-dependent intracellular growth. Taken together, our results reveal that Sey1 and LegG1 mediate LD- and FadL-dependent fatty acid metabolism of intracellularL. pneumophila.