Legionella pneumophila is an endosymbiotic bacterial species able to infect and reproduce in various protist and human hosts. Upon entry in human lungs, they may infect lung macrophages, causing Legionnaires' disease (LD), an atypical pneumonia, using similar mechanisms as in their protozoan hosts, despite the two hosts being separated by a billion years of evolution. In this study, we used experimental evolution to identify genes conferring host-specificity to L. pneumophila. To this end, we passaged L. pneumophila in two different hosts - Acanthamoeba castellanii and the human macrophage-like cells U937 - separately and by switching between the hosts twice a week for a year. In total, we identified 1518 mutations present in at least 5% of the population at the time of sampling. Half of these were localized in five groups of repeated sequences, likely to be recombination hotspots. Forty-nine mutations were fixed in the 18 populations at the end of the experiment, representing four different groups. The first two groups involve adaptation to the specific selection conditions, including (i) two specific mutations in the 30S ribosomal protein S12 (RpsL) that conferred resistance to streptomycin, to which the bacteria were unexpectedly exposed to during serial passage and (ii) two mutations, one in the 30S ribosomal protein S4 (RpsD), and one in the chaperonin GroES, both of which are likely to be fitness-restoring compensatory mutations to the original RpsL mutations. Two more interesting groups of mutations included (iii) mutations in 4 different strain-specific genes involved in LPS synthesis, found only in the lineages passaged with A. castellanii and (iv) mutations in the gene coding for LerC, a key regulator of protein effector expression, which was independently mutated in 6 lineages grown in presence of the U937 cells. We propose that the mutations degrading the function of the regulator LerC improve the fitness of L. pneumophila in human-derived cells, and that modifications in the LPS are beneficial for growth in A. castellanii. This study is a first step in further investigating determinants of host specificity in L. pneumophila.