The spontaneous ignition of biodiesel was for the first time suspected to be the cause of a recent fire in Japan. We herein present experimental evidence implying this potential risk of biodiesel. Thus, three independent biodiesel samples were subjected to a series of experiments, including a thermogravimetry-differential thermal analysis, a differential scanning calorimetry analysis and a modified wire basket test. The results were comparatively evaluated with reference to vegetable oils, of which spontaneous ignition has been well reported as a cause of fires. The heat onset temperature of biodiesel samples was determined to be ~100 °C, which was ~45 °C lower than those of vegetable oils. Furthermore, under the isothermal condition at 100 °C, the inner temperature of biodiesel samples rose rapidly with the generation of smoke after short induction periods owing to their exothermal decomposition, whereas for vegetable oils neither change in the inside temperature nor generation of smoke was observed even after 10 hours. It was therefore concluded that biodiesel possesses the higher risk of spontaneous ignition than vegetable oils. This is very important information to minimize the fire risk in biodiesel production facilities and so on. .