Mesenchymal stem cell (MSC)-based therapies are attractive strategies for multiple immune, inflammatory and degenerative diseases in dogs and humans. However, there is accumulating evidence that MSCs may promote tumor growth and progression in some settings. Cancer is generally a disease of older dogs, the population of patients likely to receive MSC therapy. Thus, an understanding of the effects of MSCs on canine cancer behavior is critical. To begin to address this, we evaluated the effects of canine bone marrow-derived MSC-conditioned medium (MSC-CM) on the growth and migration of a panel of canine tumor-derived cell lines. Compared with control medium, MSC-CM significantly promoted proliferation in 11-18 of 29 evaluated cell lines (38-62%), and proliferation was significantly inhibited in 4-7 of 29 (14-24%), depending on individual dog used for MSC-CM generation. Sarcomas were more likely to be stimulated and blood-derived tumors more likely to be suppressed by MSC-CM. MSC-CM significantly stimulated tumor cell migration in 2 of 4 evaluated cell lines (osteosarcoma and hemangiosarcoma). Demonstration that MSC-CM can exert pro-tumorigenic effects in vitro addresses a key safety concern regarding the application of MSC-based therapies in dogs with known or suspected cancer, and should inform decision making regarding MSC use in an aged dog population at risk for tumor development. At minimum, it supports the recommendation that thorough screening for cancer be undertaken prior to MSC therapy.