The p53 gene is one of the important tumour suppressor genes that are involved with the cell survival signal pathway. One of the major functions of the p53 protein is to organize cell cycle regulation and induction of apoptosis for cellular genetic stability. It has been documented that more than 50% of all human cancers include a p53 mutation. We evaluated the difference in radiosensitivity between upregulating the expression of canine wild-type p53 (cp53) in cultured osteosarcoma (D17) cells and naive D17 cells in vitro. We found that upregulating transfected cp53 D17 cells increased their radiation sensitivity in vitro, and there was a significant decrease (P < 0.009) in survival between cp53-transfected D17 cells and naive D17 cells. In this experiment, a p53 enhancement ratio (p53ER) reached approximately 3.0 at high doses. The transfected cp53 D17 cells were significantly more radiosensitive at all doses evaluated than naive D17 cells, except at 1 Gy where too few data points were available. The p53ER increased rapidly at doses less than 4 Gy, achieving a maximum of about 3.0 for doses of 4 Gy and above. This study shows the enhanced radiosensitivity of the transfected p53 at clinically relevant doses.