Updated project metadata. The facultative intracellular bacterium Legionella pneumophila employs the Icm/Dot type IV secretion system (T4SS) to replicate in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). The ER-resident large fusion GTPase Sey1/atlastin-3 promotes remodeling and expansion of LCVs, and the GTPase is also implicated in the formation of ER-derived lipid droplets (LDs). Here we reveal that LCVs intimately interact with palmitate-induced LDs in Dictyostelium discoideum amoeba. Comparative proteomics of LDs isolated from the D. discoideum parental strain Ax3 or sey1 revealed 192 differentially produced proteins, of which 7 or 22 were exclusively detected in LDs isolated from strain Ax3 or sey1, respectively. Using dually fluorescence-labeled amoeba producing the LCV marker P4C-GFP or AmtA-GFP and the LD marker mCherry-perilipin, we show that Sey1 and the L. pneumophila Icm/Dot T4SS as well as the effector LegG1 promote LCV-LD interactions. In vitro reconstitution of the LCV-LD interactions using purified LCVs and LDs from D. discoideum Ax3 or sey1 revealed that Sey1 and GTP promote this process. The LCV-LD interactions were impaired forsey1-derived LDs, suggesting that Sey1 regulates LD traits. Palmitate promoted the growth of L. pneumophila in D. discoideum Ax3 but not in sey1 and of wild-type but not mutant bacteria lacking a homologue of the E. coli fatty acid transporter FadL. Finally, isotopologue profiling indicated that intracellular L. pneumophila metabolizes 13C-palmitate, and its catabolism was reduced in D. discoideum sey1 and L. pneumophila fadL. Taken together, our results reveal that Sey1 mediates LD- and FadL-dependent fatty acid metabolism of intracellular L. pneumophila