Canine malignant melanoma is aggressive, exhibiting highly invasive and metastatic potential. Local therapies, involving surgery and/or radiation, and systemic therapies, using chemotherapy, have been used for canine melanoma treatment although canines continue to succumb to this disease. In the current studies, we verified through characterization experiments, that ICCI25S is a novel canine melanoma cell line model. Furthermore, we evaluated the radiosensitivity of canine melanoma cell lines and observed that hypofractionated radiotherapy protocols may be optimal for treatment, consistent with current clinical practice. Moreover, overactivation of the PI3K/mTOR pathway may play a role in canine melanoma radioresistance and chemoresistance. Targeting this pathway using small molecule inhibitors GSK2126458 and LY294002 decreased phosphorylated pathway proteins. Additionally, combination treatments using these inhibitors with carboplatin were more efficacious at decreasing melanoma cell proliferation compared to using any agent alone. Inhibition of the PI3K/mTOR pathway may be worthy of further investigation for systemic canine melanoma treatment.