ABSTRACT Legionella pneumophila is a natural parasite of environmental amoebae and the causative agent of a severe pneumonia termed Legionnaires' disease. The facultative intracellular pathogen employs a bipartite metabolism, where the amino acid serine serves as the major energy supply, while glycerol and glucose are mainly utilized for anabolic processes. The L. pneumophila genome harbors the cluster lpg1653 to lpg1649 putatively involved in the metabolism of the abundant carbohydrate myo -inositol (here termed inositol). To assess inositol metabolism by L. pneumophila , we constructed defined mutant strains lacking lpg1653 or lpg1652 , which are predicted to encode the inositol transporter IolT or the inositol-2-dehydrogenase IolG, respectively. The mutant strains were not impaired for growth in complex or defined minimal media, and inositol did not promote extracellular growth. However, upon coinfection of Acanthamoeba castellanii , the mutants were outcompeted by the parental strain, indicating that the intracellular inositol metabolism confers a fitness advantage to the pathogen. Indeed, inositol added to L. pneumophila -infected amoebae or macrophages promoted intracellular growth of the parental strain, but not of the Δ iolT or Δ iolG mutant, and growth stimulation by inositol was restored by complementation of the mutant strains. The expression of the P _iol promoter and bacterial uptake of inositol required the alternative sigma factor RpoS, a key virulence regulator of L. pneumophila . Finally, the parental strain and Δ iolG mutant bacteria but not the Δ iolT mutant strain accumulated [U- ¹⁴ C ₆ ]inositol, indicating that IolT indeed functions as an inositol transporter. Taken together, intracellular L. pneumophila metabolizes inositol through the iol gene products, thus promoting the growth and virulence of the pathogen. IMPORTANCE The environmental bacterium Legionella pneumophila is the causative agent of a severe pneumonia termed Legionnaires' disease. The opportunistic pathogen replicates in protozoan and mammalian phagocytes in a unique vacuole. Amino acids are thought to represent the prime source of carbon and energy for L. pneumophila . However, genome, transcriptome, and proteome studies indicate that the pathogen not only utilizes amino acids as carbon sources but possesses broader metabolic capacities. In this study, we analyzed the metabolism of inositol by extra- and intracellularly growing L. pneumophila . By using genetic, biochemical, and cell biological approaches, we found that L. pneumophila accumulates and metabolizes inositol through the iol gene products, thus promoting the intracellular growth, virulence, and fitness of the pathogen. Our study significantly contributes to an understanding of the intracellular niche of a human pathogen.