The aim of this study was the identification of proteins differentially represented in the serum proteome of seropositive dogs with (Group 1) and without (Group 2) clinical-pathologic signs consistent with ehrlichiosis compared to healthy control dogs. Serum samples were collected from 20 dogs of various breeds with naturally occurring ehrlichiosis (10 dogs belonged to Group 1 and 10 to Group 2) and 10 healthy dogs. Two-dimensional electrophoresis (2DE) of pooled serum for each of the group of dogs were run in triplicate. 2D image analysis showed 39 spots differently expressed between Group 1 and Group 2 compared with healthy ones. Mass spectrometry analysis allowed identification of 6 proteins: albumin, haptoglobin (Hp), alpha-1-antitrypsin (AAT), Retinol Binding Protein 4 (RBP-4), alpha-1-acid glycoprotein (AGP) and vitamin D-binding protein (VDBP). When a confirmatory study was performed for albumin, Hp, AAT and RBP-4 by using different assays, significant differences (P < 0.05) between diseased and healthy groups were observed. It can be concluded that there are significant changes in the serum proteome of dogs with ehrlichiosis with modifications in proteins related with the acute phase response such as Hp, albumin and AGP, with vitamin A transport such as RBP-4, with inhibitors of serine proteases and anti-inflammatory proteins such as AAT, and vitamin D metabolism and actin scavengers such as VDBP.