The purpose of this study was to clarify the mechanism underlying the therapeutic effects of radiofrequency hyperthermia in naturally occurring OA of Hartley guinea pigs by investigating the level of autophagy. Thermotron-RF300, which can apply a 13.56 MHz radio frequency pulse to the animal knees was used to perform hyperthermia. The quantitative analysis of the distribution of positive cells showed that, in the normal cartilage, ULK1 and Beclin1 were highly expressed. Seven days after the exposure to the radiofrequency, the expression levels of ULK1 and Beclin1 were augmented in animals with moderate OA. The severity of cartilage degradation was significantly reduced in the radiofrequency-treated knees compared with the untreated left knees. These results suggest that radiofrequency hyperthermia can stimulate autophagy, which is reduced in OA cartilage. In addition, radiofrequency reduced the severity of spontaneous osteoarthritis.