Osteosarcoma is a mesenchymal malignant bone tumor accompanied by a high rate of lung metastasis and short survival in dogs. Although various therapies have been reported, the etiological mechanism of osteosarcoma remains undetermined and the development of novel therapeutic agents is warranted. In this study, we have reported the diverse functions of quercetin, one of the well‐known flavonoid, in D‐17 and DSN (canine osteosarcoma) cell lines. Current results indicate that quercetin decreases proliferative properties and increases programmed cell death, in addition to altering the cell cycle, mitochondrial depolarization, level of reactive oxygen species, and concentration of cytoplasmic calcium in both cells. Furthermore, it was observed that quercetin suppresses phosphorylation of AKT, P70S6K, and S6 proteins and upregulates phosphorylation of ERK1 or 2, P38, c‐Jun N‐terminal kinase, and P90RSK proteins in both cell lines. Collectively, we suggest that quercetin can be used as a pharmacological agent for suppressing the proliferation and inducing the apoptosis of canine osteosarcoma cells.