Legionella pneumophilaproliferates in environmental amoeba and human cells within the Legionella-containing vacuole (LCV). The exported AnkB F-box effector ofL. pneumophilais anchored into the LCV membrane by host-mediated farnesylation. Here, we report that host proteasomal degradation of Lys48-linked polyubiquitinated proteins, assembled on the LCV by AnkB, generates amino acids required for intracellular bacterial proliferation. The severe defect of theankBnull mutant in proliferation within amoeba and human cells is rescued by supplementation of a mixture of amino acids or cysteine, serine, pyruvate, or citrate, similar to rescue by genetic complementation. Defect of theankBmutant in intrapulmonary proliferation in mice is rescued upon injection of a mixture of amino acids or cysteine. Therefore,Legionellapromotes eukaryotic proteasomal degradation to generate amino acids needed as carbon and energy sources for bacterial proliferation within evolutionarily distant hosts.