Updated publication reference for PubMed record(s): 26678042. Mycoplasma suis belongs to the hemotrophic mycoplasma (HM) that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture HMs in vitro has hindered their characterization at the molecular level. Although genome sequences of eight HMs are available, there is only one study based on genome sequenced data on the proteomic level for HMs, namely M. suis. In the present work, the proteome of M. suis strain KI_3806 during acute infection was extended significantly by applying three different protein extraction methods, 1D SDS-PAGE and LC-MS/MS. A total of 404 of the 795 M. suis KI_3806 proteins (50.8%) of all encoded proteins were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane-associated hypothetical proteins that might be involved in adhesion or host-pathogen interaction. Furthermore, analyses of the expressed transporters indicated the existence of a hexose-6-phosphate-transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture.