Curcuma longa L., also known as turmeric, has been widely studied for its various therapeutic properties, including antineoplastic action. The ethanolic extract of the plant contains several phenolic compounds, especially curcumin. Osteosarcoma is a predominant bone tumor in dogs and humans, characterized by high metastatic potential and an unfavorable prognosis. The aim of this study was to investigate the effects of turmeric ethanol extract on canine osteosarcoma cells from established culture. The cells were cultured and treated with different curcumin concentrations (0, 10 μM, 20 μM, 50 μM, 100 μM, and 1000 μM) and exposure times (24h, 48h, and 72h). We first performed tetrazolium reduction technique (MTT) assay and calculated IC50. An immunocytochemistry assay was performed after extract treatment to verify the expression of mutated p53 and therefore study the proliferative potential of malignant cells; Bcl-2 and Ki-67 were used to assess apoptosis and the degree of malignancy, respectively. The extract enhanced the proliferation of canine osteosarcoma cells, reaching 3,819.74% at 50 μM of curcumin. The extract also significantly altered the expression of mutated p53 and Ki-67 proteins but not that of Bcl-2, suggesting that it did not induce this antiapoptotic pathway. Overall, these results are prerequisite to better understanding how natural compounds such as turmeric ethanolic extract affect cell proliferation and could be used to treat various diseases.