<p>Radio-resistance of cancer stem-like cells (CSCs) is associated with the failure of radiation therapy. ZsGreen1-positive (ZsG⁺) cells, which exhibit low proteasome activity, have been used to enable the detection and isolation of CSCs. However, the mechanisms of radio-resistance in canine tumor cells with low proteasome activity remain unclear. This study aimed to elucidate the radio-resistance mechanisms of ZsG⁺ cells by identifying a potential target of canine CSCs. ZsG⁺ cells, isolated using flow cytometric cell sorting, were compared with ZsG⁻ cells. Sulfasalazine was used to suppress glutathione (GSH) synthesis by inhibiting xCT. In vitro experiments demonstrated a significantly higher radio-resistance in ZsG⁺ cells than in ZsG⁻ cells. After X-irradiation, ZsG⁺ cells had fewer p53‑binding protein 1 (53BP1) foci, low reactive oxygen species (ROS) accumulation, and high GSH content. Sulfasalazine caused radiosensitization of ZsG⁺ cells with an increased number of 53BP1 foci by decreasing GSH contents and increasing ROS accumulation. The low proteasome activity played a role in xCT upregulation. In conclusion, canine tumor cells with low proteasome activity are radio-resistant due to high GSH content and low ROS accumulation. Sulfasalazine causes radiosensitization of the tumor cells by altering redox balance by inhibiting GSH synthesis for effective targeting of canine CSCs.</p>