Babesiosis is a disease of significant medically and veterinary importance with worldwide distribution. It is caused by intra-erythrocyte protozoal parasites, withBabesia rossicausing the most severe clinical signs of all the largeBabesiaparasites infecting dogs. The disease can be clinically classified into uncomplicated and complicated forms with a wide range of clinical presentations from a mild, subclinical illness to complicated forms and death. The aim of this study was to assess serum proteomic profiles from dogs with babesiosis and healthy dogs using a label-based proteomics approach. Altogether 32 dogs naturally infected withB. rossi(subdivided into 18 uncomplicated cases and 14 complicated cases of babesiosis) and 20 healthy dogs were included. There were 78 proteins with significantly different abundances between the three groups of dogs. Elucidation of proteins and pathways involved in canine babesiosis caused byB. rossihave revealed key differences associated with haemostasis, innate immune system, lipid metabolism and inflammation. Shotgun proteomic profiling allowed identification of potential serum biomarkers for differentiation of disease severity in canine babesiosis caused byB. rossi. These findings may be applicable to the study of host-parasite interactions and the development of novel therapeutic targets.