The adult stem cells have become prominent candidates for the treatment of various diseases in veterinary practice. Therefore, the main goal of our study was to provide a complex study of canine bone marrow derived mesenchymal stem cells (BMMSCs) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols the multilineage differentiation and specific surface markers of MSCs were supplemented with their gene expression and the proteome profile. Present data has confirmed that canine BMMSCs expresses important genes for differentiation toward osteo-, chondro- and tendo-genic directions, but also genes that were associated with neurotrophic and immunomodulatory properties. Furthermore, we have identified for the first time by proteome profiling dynamic release various bioactive molecules, such as transcription and translation factors, osteogenic, growth and neurotrophic factors from canine BMMSC conditioned medium. Importantly, relevant genes were linked to their proteins that were detected in conditioned medium, which have an important correlation to their possible function and clinical application. Thus, here we show that the canine BMMSCs properties are applicable for replacement therapies of skeletal disease, but due to their ability of releasing immunomodulatory and bioactive molecules via conditioned media, they may improve repair of other tissue. However, extensive experimental or pre-clinical trials testing canine sources needs to be performed in order to better understand the individual differences between healthy donors that are considered for transplantation treatment studies in veterinary medicine.