Updated project metadata. Surface proteome of microbial pathogens is a crucial component of host-pathogen interactions. We applied cell surface biotinylation in association with high-throughput proteomic analysis to identify and quantify the surface proteome of a virulent and an attenuated strain, originating from the same single cell of H. meleagridis. In total 1485 proteins were identified amongst the two strains. Quantification revealed 22 and 45 up-regulated proteins in the virulent and in the attenuated strain, respectively. In the virulent strain proteins such as alpha-amylase, Clan CD family C13 asparaginyl endopeptidase-like cysteine peptidase, two Clan SC family S33, methylesterase-like serine peptidases, LysM peptidoglycan-binding domain-containing protein and surfactant B were up-regulated and could be linked to putative virulence factors involved in the colonization of the host and establishment of an infection. In the attenuated strain structural proteins such as actin-related proteins, fimbrin, coronin, were up-regulated, alongside with various transport and energy production proteins. Our results speak for the protozoan’s adaptation to the in vitro environment, with a clear tendency for up-regulation of mainly metabolic and structural proteins. We present a comprehensive profiling of H. meleagridis surface proteome. These results provide not only a better understanding of the surface molecules that may participate in establishing and maintaining infection but also highlights the pathogen’s in vitro adaptation processes.