Legionella pneumophila proliferates in environmental amoeba and human cells within the Legionella-containing vacuole (LCV). The exported AnkB F-box effector of L. pneumophila is anchored into the LCV membrane by host-mediated farnesylation. Here, we report that host proteasomal degradation of Lys 48-linked polyubiquitinated proteins, assembled on the LCV by AnkB, generates amino acids required for intracellular bacterial proliferation. The severe defect of the ankB null 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 the ankB mutant in intrapulmonary proliferation in mice is rescued upon injection of a mixture of amino acids or cysteine. Therefore, Legionella promotes eukaryotic proteasomal degradation to generate amino acids needed as carbon and energy sources for bacterial proliferation within evolutionarily distant hosts.