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Canine osteosarcoma (OSA) is the most common form of bone cancer diagnosticated in dogs and is highly metastatic. There has been limited advancement in discovering an effective treatment for OSA in the last few decades. The major drawback of the currently used chemotherapeutic drugs is their side effects. In this preliminary study, we investigated the efficacy of using a novel food-derived drug, phloridzin docosahexaenoate (PZ-DHA), in the treatment of canine OSA in vitro. PZ-DHA was selectively cytotoxic to canine OSA D17 cells, while normal human liver cells (WRL68) were more resistant. We also found that PZ-DHA had enhanced cellular uptake in D17 cells compared to its precursors and in WRL68 cell line. This study provides preliminary evidence that PZ-DHA needs to be further assessed as a safe and efficacious new drug in the treatment of both canine and human OSA.