BACKGROUND: The demethylating agent decitabine (DAC) effectively inhibits tumor growth and metastasis by targeting ESR1 methylation to restore estrogen receptor alpha (ERα) signaling and promoting cellular differentiation in models of human osteosarcoma (OSA). Whether this pathway can be targeted in canine OSA patients is unknown. METHODS: Canine OSA tumor samples were tested for ERα expression and ESR1 promoter methylation. Human (MG63.3) and canine (MC-KOS) OSA cell lines and murine xenografts were treated with DAC and , respectively. Samples were assessed using mRNA sequencing and tissue immunohistochemistry. RESULTS: ESR1 is methylated in a subset of canine OSA patient samples and the MC-KOS cell line. DAC treatment led to enhanced differentiation as demonstrated by increased ALPL expression, and suppressed tumor growth and . Metastatic progression was inhibited, particularly in the MG63.3 model, which expresses higher levels of DNA methyltransferases DNMT1 and 3B. DAC treatment induced significant alterations in immune response and cell cycle pathways. CONCLUSION: DAC treatment activates ERα signaling, promotes bone differentiation, and inhibits tumor growth and metastasis in human and canine OSA. Additional DAC-altered pathways and species- or individual-specific differences in DNMT expression may also play a role in DAC treatment of OSA.