The hot spot temperature inside the windings of the oil-immersed power transformers may exceed 200 °C due to some winding deficiencies, posing negative impact on the paper insulation. A series of heating experiments are deliberately conducted with oil-paper insulation samples to study the influence of the hot spot temperature ranging from 120 to 280 °C, and the insulation degradation is characterized by the breakdown voltage (BDV) and the degree of polymerization (DP). Furthermore, surface morphology, water content and cellulose degradation mechanism are analyzed so as to reveal the causes of the above phenomena. The experimental results indicate that with increase of the hot spot temperature, the breakdown voltage increases after the first drop, while the DP continues to decline in the tested temperature range. No obvious linear relationship is observed between the hot spot temperature and the DP or the BDV. Regarding the test samples the authors have studied, the inflection point of the hot spot temperature for the insulation deterioration is found to be between 240 and 260 °C. The proposed research presents a fundamental reference for assessment of the winding insulation degradation from the viewpoint of hot spot temperature.