Background: Acquiring the subcellular localization of proteins in Mycoplasma hyopneumoniae(M.hyopneumoniae),the etiological agent of porcine enzootic pneumonia,is helpful for understanding the pathogenic mechanism and searching for candidate protective antigens for a more effective vaccine.Although some proteins' localizations have been identified experimentally or annotated in genomic data in previous studies,most of the proteins' localizations are unknown or unclear.Results: We predicted and manually curated the subcellular localization of proteins in M.hyopneumoniae strain 232 by following a conducting workflow and some criteria that incorporated computational predictions of signal peptides,secretion systems,transmembrane domains,protein domain content,experimental data,etc.Meanwhile,the conservation of proteins was analyzed by using all five genome-sequenced and annotated M.hyopneumoniae strains.With comprehensive prediction,data incorporation and manual curation,we concluded that M.hyopneumoniae contains 650 conserved proteins,including 390 cytoplasmic proteins,168 inner membrane proteins,23 periplasmic proteins,136 outer membrane proteins and 3 extracellular proteins(some proteins have multiple localizations).Computational predictions,conducted at the M.hyopneumoniae genome scale,provide less costly localization information,which might not be readily derived from functional annotations but solely from sequence similarity.Conclusions: The localization prediction of all of the M.hyopneumoniae proteins is not simply a matter of developing bioinformatics information.It will also be useful for localization identification experiments,pathogenic mechanism studies,more effective antigenic candidate protein discoveries,and new vaccine development.